Treatment composition comprising physically disrupted tooth pulp and non-cultured stem cells in a matrix

ABSTRACT

A medical implant comprising in components from a tooth and stem cells harvested from at least one tooth. Tooth stem cells may be harvested from the dental pulp of mammalian teeth, such as unerupted third molars in humans. After the stem cells are removed and isolated from the other teeth tissue, the hard tooth may be ground into a base material for the manufacture of a porous matrix into which the tooth stem cells can be added. Additionally, soft tissue from the harvested tooth may be used as a carrier scaffold for soft tissue applications such as meniscal or cartilage repair.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/655,633, filed on Jan. 4, 2010, now U.S. Pat. No. 8,470,308 which isa continuation-in-part of, and claims priority to, U.S. patentapplication Ser. No. 12/348,280, filed on Jan. 3, 2009, now abandonedthe disclosures of each of which are hereby incorporated by reference.

RELATED ART

1. Field of the Invention

The present disclosure relates to harvesting stem cells from teeth andthereafter using the harvested stem cells and parts of the tooth, suchas soft tissue, to create medical implants that are compatible with themammalian body.

2. Brief Discussion of Related Art

Scientific Progress and Future Research Directions (June 2001), indicatethat sources of stem cells include bone marrow, peripheral blood, bloodvessels, the cornea and the retina of the eye, brain, skeletal muscle,dental pulp, liver, skin, the lining of the gastrointestinal tract, andpancreas. Methods and apparatus have been developed to remove stem cellsfrom some of these areas of the mammalian body. As to removal of stemcells from bone material specifically, such methods and apparatusinclude U.S. Patent Publication No. 2008/0176325 to Bowermaster, et al.,the contents of which are incorporated herein by reference.

INTRODUCTION TO THE INVENTION

Referring to FIGS. 1 and 3, harvesting dental pulp from a tooth 100requires dissecting or opening the tooth to reveal the molar pulp andstem cells 124, or otherwise manipulating the pulp to remove it from thepulp chamber 120 and root canal 122. The pulp can be separated into asoft tissue connective tissue 134 and stem cells 136. Thereafter, thestem cells 136 may be combined with one or more of the following:processed soft tissue 134 (shown in test tubes); ground hard aspects 138of the harvested tooth 100; other autologous patient tissues; autologousblood concentrates; growth factors; autologous biologic bone particlesor a matrix; and a synthetic biocompatible graft material or scaffold.In this respect, this disclosure provides a means for harvesting stemcells from the pulp of teeth (and other cells and tissues which may befound in the removed tooth) and utilizing these harvested materials formedical objectives. Such medical objectives include, without limitation,fostering or accelerating bone ingrowth and joint repair, reconstructionor reconstitution. Such medical devices also include fostering oraccelerating connective tissue regrowth and repair, and fostering oraccelerating non-connective tissue regrowth and repair.

Pursuant to the instant disclosure, orthopedic implants may be createdthat have true bone ingrowth capabilities, thereby obviating the needfor cement or other artificial adhesion techniques. Other exemplary bonerepair, reconstructive, and regeneration uses within the scope of theinstant disclosure include fracture treatment, fracture non-uniontreatment, bone fusions, bone lengthenings, bone defect repair, boneosteotomies and other orthopedic, maxillofacial, orthodontic human andveterinary applications.

In addition to these bone uses for repair, reconstruction, andreconstitution, the products and implants of the present disclosure maybe used in combination with tooth collagen and tooth connective tissuefor soft tissue and joint applications. Dental pulp stem cells combinedwith tooth tissue collagen and connective tissue may be utilized for avariety of clinical purposes including wound treatment, hemostasis,nerve repair, joint cartilage and meniscal repair, ligament and tendonand soft tissue augmentation, repair and reconstruction. Soluble stemcell collagen and tooth soft tissue products may be used as asubcutaneous implant for repairing dermatological defects such as acnescars, glabellar furrows, excised scars and other soft tissue defects.

The present disclosure also provides bone or soft tissue ingrowththrough cell migration into the interstices of a tooth derived collagenor connective tissue 142 or ground tooth particulate matrix 144. Inexemplary form, the tooth derived collagen 142 or ground toothparticulate matrix 144 may have varying degrees of porosity to create askeleton providing sufficient interstices and volume for cells to attachand grow into the matrix, and to synthesize their own macromolecules.These attached cells then produce the desired new matrixcharacterizations, which allows for the growth of additional new tissuefor the particular, specific application.

Additives such as hyaluronic acid and fibronectin may augment the softtissue implants of the present disclosure. Hyaluronic acid in a collagenmatrix, for example, encourages cellular infiltration into the pores andchannels of the matrix. Fibronectin, on the other hand, induces cellattachment to fibers of a collagen matrix, for example. Autologous andallogenous blood concentrates and tissue derivatives 154 can also beadded to the dental pulp derived products to enhance their function,viability, and incorporation.

In a first exemplary embodiment, the present disclosure relatesgenerally to a composition comprising a collection of pluripotent stemcells collected from the dental pulp of a harvested human tooth (such asa third molar), whether or not the stem cells have been duplicated orpurified (i.e., cell passes), in combination with at least a portion ofa processed mammalian tooth (hard and/or soft components of the tooth,such as enamel and connective soft tissue within the tooth and pulpsegments).

In a second exemplary embodiment, the present disclosure relatesgenerally to a medical implant composition comprising a collection ofpluripotent stem cells collected from the dental pulp of a harvestedhuman tooth, whether or not the stem cells have been duplicated orpurified (i.e., cell passes), in combination with at least a portion ofa mammalian tooth and a synthetic particulate or synthetic matrix formedcomprising at least one of a porous biocompatible metal (porous tantalumand porous titanium), a biocompatible thermoplastic, a biocompatiblethermoset, and a ceramic based biocompatible mineral (Demineralized bonematrix, calcium phosphates (e.g., Hydroxyapatite (HA) and β-tricalciumphosphate) calcium sulfate composites (e.g., OsteoGraf [DENTSPLYFriadent CeraMed, Lakewood, Colo.], Norian SRS [Synthes, Inc, WestChester, Pa.], ProOsteon [Interpore Cross International, Irvine,Calif.], Osteoset [Wright Medical Technology, Inc, Arlington, Tenn.])and degradable and nondegradable polymer-based bone graft substitutes,(e.g., Cortoss [Orthovita, Inc, Malvern, Pa.], open porosity polylacticacid polymer [OPLA], Immix [Osteobiologics, Inc, San Antonio, Tex.]).

In a third exemplary embodiment, the present disclosure relatesgenerally to a bone graft slurry comprising a collection of pluripotentstem cells collected from the dental pulp of a harvested human tooth,whether or not the stem cells have been duplicated or purified (i.e.,cell passes), in combination with at least a portion of a ground,particulate mammalian tooth.

In a fourth exemplary embodiment, the present disclosure relatesgenerally to a collagen slurry comprising a collection of pluripotentstem cells collected from the dental pulp of a harvested human tooth,whether or not the stem cells have been duplicated or purified (i.e.,cell passes), in combination with at least a portion of a soft tissuecomponent from the mammalian tooth papilla.

In a fifth exemplary embodiment, the present disclosure relatesgenerally to a method of forming a biologic matrix implant, comprisingthe steps of: processing at least a portion of a mammalian tooth to formparticulate matrix; combining the particulates from the mammalian toothwith a collection of pluripotent stem cells collected from the dentalpulp of a harvested human tooth, whether or not the stem cells have beenduplicated or purified (i.e., cell passes), to form a biologic matriximplant.

In a sixth exemplary embodiment, the present disclosure relatesgenerally to a method of forming a biologic matrix implant, comprisingthe steps of: processing at least a portion of a mammalian tooth to formparticulates; combining the particulates from the mammalian tooth with asource of hydroxyapatite to form a biologic matrix; and, combining thebiologic matrix with a collection of pluripotent stem cells collectedfrom the dental pulp of a harvested human tooth, whether or not the stemcells have been duplicated or purified (i.e., cell passes), to form abiologic matrix implant.

In a seventh exemplary embodiment, the present disclosure relatesgenerally to a method of forming a biologic matrix, comprising the stepsof: processing at least a portion of a mammalian tooth to particulates;and, combining the particulates from the mammalian tooth with a sourceof hydroxyapatite to form a biologic matrix.

In an eighth exemplary embodiment, the present disclosure relatesgenerally to a soft tissue composition of matter comprising: acollection of pluripotent stem cells collected from the dental pulp of aharvested human tooth, whether or not the stem cells have beenduplicated or purified (i.e., cell passes); and, connective tissueharvested from the dental pulp of a harvested human tooth.

In a ninth exemplary embodiment, the present disclosure relatesgenerally to a method of forming a soft tissue replacement comprising:collecting pluripotent stem cells from dental pulp of a harvested humantooth, whether or not the stem cells have been duplicated or purified(i.e., cell passes); collecting connective tissue harvested from thedental pulp of a harvested human tooth; and, combining the harvestedconnective tissue with the harvested pluripotent stem cells to form anHLA matched soft tissue replacement.

In a tenth exemplary embodiment, the present disclosure relatesgenerally to a medical implant composition comprising: a collection ofpluripotent stem cells collected from the dental pulp of a harvestedhuman tooth; at least a portion of a mammalian tooth; and, an autologousbiological matrix formed from a source selected from the groupconsisting of the harvested tooth, bone from an autologous donor, andsoft tissue from the donor.

In an eleventh exemplary embodiment, the present disclosure relates to amethod of making an HLA ABO matched or unmatched allograft medicalimplant, comprising: removing a human tooth from a donor; harvestingpluripotent cells from the pulp of the tooth; cryogenically preservingthe pluripotent cells and the tooth; pulverizing the tooth to form abone powder therefrom; forming a bone matrix from the tooth powder; and,combining the pluripotent cells and the matrix to form an allograftmedical implant.

In a twelfth exemplary embodiment, the present disclosure furtherrelates to a method of fabricating a medical implant, comprising:removing a human tooth from a donor; harvesting pluripotent cells fromthe pulp of the tooth; cryogenically preserving at least one of thepluripotent cells and the tooth; pulverizing the tooth to form a bonepowder therefrom; forming an autologous bone particulate matrix from thetooth powder; and, combining the pluripotent cells and the particulatematrix to form the implant.

In a thirteenth exemplary embodiment, the present disclosure furtherrelates to a method of fabricating a soft tissue medical implant,comprising: removing a human tooth from a donor; harvesting pluripotentcells from the pulp of the tooth; cryogenically preserving thepluripotent cells and the tooth; separating the pulp stem cells from thesurrounding collagen and soft tissue; forming an autologous collagen orconnective tissue matrix from these tooth tissues; and, combining thepluripotent cells and the matrix to form the soft tissue medicalimplant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the sequence of utilized totransform a harvested tooth into a multitude of intermediates (stemcells, mineral and soft-tissue components) and thereafter a multitude ofproducts.

FIG. 2 includes a series of drawings showing tooth hydroxyapatite (THA)graft particulate, a tooth and collagen and soft connective tissuestandard shaped scaffolds and matrixes, and various shaped collagen andTHA scaffold and matrixes for use with the exemplary embodiments of theinstant disclosure.

FIG. 3 is a cross section of a mammalian molar tooth taken vertically.

FIG. 4 is a rear view of a human spinal fusion device showing the spinalfusion bracket instrumentation in position.

FIG. 5 is a rear view of a human spinal fusion making use of a stem cellmolar graft bone slurry located proximate the intended bone fusionlocations.

FIGS. 6-8 include a series of figures showing human bony non-unions andhow the non-unions are treated with stem cell molar bone graft slurryand THA, and thereafter enclosed, to facilitate bone formation at thenon-union site.

FIGS. 9-11 include a series of figures showing a human scaphoid fractureand how the scaphoid fracture is treated with stem cell molar bone graftslurry and THA, and thereafter enclosed, to facilitate bone formation atthe fracture site.

FIG. 12 is a top, plan view of a human vertebrae being injected withstem cell molar bone graft slurry to treat vertebral fractures andosteoporosis.

FIG. 13 is a frontal view of an Ilizarov device distracting an osteotomesite of the lower leg of a human, where the osteotome site is augmentedwith stem cell molar bone graft slurry.

FIG. 14 is a frontal view of human lower leg bones, the fibula andtibia.

FIG. 15 is a profile view showing the general injection and bonding sitefor a stem cell slurry on a prosthetic tibial tray having a porous stem.

FIG. 16 is a frontal view of human lower leg bones, the fibula andtibia, where a total knee arthroplasty procedure has been carried outusing a cementless femoral and patella components, and the cementlesstibial tray of FIG. 15 implanted into the tibia.

FIG. 17 is a profile view of a human skull showing physical changesresulting from a sinus lift procedure and a distraction osteogenesisprocedure, both using stem cell molar bone graft slurries in accordancewith the instant disclosure.

FIGS. 18-20 include a series of figures showing a human cleft palate andhow the cleft palate is treated with stem cell molar bone graft slurryand custom 3-D matrix, and thereafter enclosed, to facilitate boneformation at the cleft palate site.

FIG. 21 is a profile view of a human lower leg showing certain bones,muscles, and a partially torn Achilles tendon.

FIG. 22 is a profile view of a human lower leg showing certain bones,muscles, and a torn Achilles tendon, where the torn Achilles tendon istreated with a stem cell soft tissue slurry and pulp soft tissue derivedmatrix formulated in accordance with the instant disclosure.

FIG. 23 is a profile view of an equine lower leg showing bones, muscles,and connective tissues, where stem cell slurries were specificallyformulated for certain purposes (e.g., bone, soft tissue, connectivetissue, etc.) and injected to treat certain ailments.

FIG. 24 is frontal view of a canine pelvis, where stem cell slurrieswere specifically formulated for certain purposes (e.g., bone, softtissue, connective tissue, nerves, etc.) and injected to treat certainailments.

DETAILED DESCRIPTION

The exemplary embodiments of the present disclosure are described andillustrated below to encompass harvesting stem cells from teeth andthereafter using the stem cells in addition to other tooth material tocreate medical implants and therapeutic compositions such as tissuegrowth accelerators or regeneration formulations that are compatiblewith the mammalian body. Of course, it will be apparent to those ofordinary skill in the art that the exemplary embodiments discussed belowmay be reconfigured without departing from the scope and spirit of thepresent disclosure. However, for clarity and precision, the exemplaryembodiments as discussed below may include optional steps, methods, andfeatures that one of ordinary skill should recognize as not being arequisite to fall within the scope of the present disclosure. Forexample, the devices and methods disclosed are applicable to all mannerof medical implants anatomies, including spine, knee, hip, shoulder,elbow, skull, maxilla, and the like.

Referencing FIG. 3, an erupted human tooth 100 anatomically includes acrown 110, a neck 112, a root 114, enamel 116, dentin 118, a pulp cavity120, and a root canal 122. Pluripotent stem cells may be located withinthe pulp cavity 120 of a mammalian tooth, specifically the pulp cavity120 of unerupted wisdom teeth (see FIG. 17, numeral 1732). An exemplarymethod of harvesting stem cells from molar teeth as disclosed in U.S.Patent Publication 20080176325 to Bowermaster, et al., the disclosure ofwhich is incorporated herein.

The primary difficulties encountered with harvesting stem cells fromsources other than dental pulp has been a more limited number of cellsand relatively high degree of difficulty culturing and growing theisolated cells. In contrast, molar dental pulp normally containsmillions of stem cells. These cells are relatively homogeneous andexhibit markers associated with embryonic stem cells. In addition tostem cells, the harvested molar tooth pulp also includes connectivetissue cells that are relatively easy to culture and reproduce.

Stem cells can be readily obtained by isolating the developing dentalpulp or mesenchymal dental papilla from an unerupted tooth bud such as,for example, an unerupted third molar. A tooth bud is a knoblikeprimordium that develops into an enamel organ surrounded by a dentalsac, encasing the dental papilla. Dental papilla is a mass ofmesenchymal tissue that ultimately differentiates to form dentin anddental pulp. The dental sac ultimately differentiates to form theperiodontal ligament. For each unerupted tooth, a tissue mass can beisolated to provide two to five million stem cells. As used herein,“isolated” or “isolating” refers to removal of the tissue masscontaining stem cells from the oral cavity of a mammal, including ahuman.

Tooth buds appear in early childhood, with the last, the third molar,beginning to form at approximately four years of age in a human. By thetime the twenty deciduous teeth have erupted in a human, the firstpermanent molars have also erupted or are erupting. There areapproximately twenty-eight tooth buds for permanent teeth in variousstates of development in the tissue beneath the deciduous teeth. By thetime the molar teeth erupt, the enamel organ has generally encased thedental pulp. Prior to eruption, however, the mesenchymal tissue may besurgically removed to provide an isolated tissue comprising millions ofstem cells. It is to be understood, however, that any tooth bud orunerupted mammalian tooth may provide an isolated tissue for extractionof stem cells according to the present disclosure.

As mentioned previously, an attractive source of isolated tissue is anunerupted third molar. This is particularly the case since thisdeveloping tooth is often surgically removed without negativeconsequences to the patient. In addition, it is known that stem cellsfrom third molars may differentiate into osteoblasts (Osteogenicpotential molar stem cells alone, J. Oral Maxillofac Surg. 2009 March,67(3):501-6; Evaluation of pluripotency in human dental pulp cells,Koyama N, et al., Pharmacogenomics J., 2009 Sep. 1, PMID:19721467;Isolation and characterization of stem cells derived from human thirdmolar tooth germs of young adults: implications in neo-vascularization,osteo-, adipo- and neurogenesis, Yalvac M E, et al, The PharmacogenomicsJournal advance online publication, 2009 Sep. 1).

Third molars, often called “wisdom teeth,” generally erupt between theages of 17 and 21 in a human. Second molars usually erupt between theages of 11 and 13 in a human, but allow during this time frame detectionof developing third molars by x-ray or other imaging devices. If thereis insufficient room for the third molar or it is developing abnormally(e.g., some third molars appear to be growing “sideways” in maxilla ormandible), the third molar may be surgically removed before it becomesimpacted (which may occur if the developed tooth has not reached itsappropriate final position by adulthood) or causes misalignment of otherteeth.

Third molars are customarily removed from pre-teen and teenage patientswhile the molars are still developing, and while the primordial bulbstill contains millions of stem cells. Third molars may be extractedfrom living humans, as well as juveniles that are deceased proximate thetime of death. In particular, just as an organ transplant donor mayprovide viable organs proximate the time of death, the same donor(presuming a juvenile) may provide one to four third molars from whichstem cells may be harvested consistent with the instant disclosure.Hydroxyapatite (HA), having a chemical formula of Ca₁₀(PO₄)₆(OH)₂, isthe main mineral constituent of human bone and human teeth, and is anoutstanding bone substitute because of its osteoconductive properties.

Human Derived Tooth Hydroxyapatite (THA) may be used as a bone graftsubstitute. In ashed or calcinated form, the natural bioceramic THA isvery successful in promoting osteointegration. THA is also a safebone-graft bioceramic material for bone grafting. THA can be formed intopowders and particulate, just like other sources of hydroxyapatitecurrently being used (e.g., HA ceramics can be manufactured from naturalmaterials such as coral to create scaffolds or matrices. Any of theseTHA derived forms allow for enhanced osteointegration when combined withthe associated dental pulp stem cells.

Teeth, such as third molars, that are discarded during the normal dentalcycle could also be stored or cryopreserved for later combination withthird molar THA to increase the amount a graft material available. Sinceapproximately 800,000 third molars (generally the last set of teeth toerupt) are removed each year in the United States alone, the number ofavailable stem cells is not insignificant. Removal of four third molarsfrom a single donor can easily yield eight to over twenty millionmultipotent (i.e., pluripotent) stem cells. A majority of these stemcells harvested from teeth, particularly third molars, are Oct4positive, SSEA1 positive, SCA1 negative, MART-1 negative, TRA80-1positive, SSEA-4 negative, CD117 negative and TRA60-1 negative,indicating that the stem cells are primitive, multipotent cells that maybe induced to differentiate into a variety of cell and tissue types.

In a preferred circumstance, one or more teeth are extracted from theintended recipient of the medical implant or therapeutic stem cellcompositions in order to create an autologous dental pulp stem cellproduct. In such a circumstance, the stem cell donor and bone or softtissue graft patient are the same person. However, siblings and otherindividuals closely related to the intended recipient (sometimesreferred to herein as the “patient”) in need of dental pulp stem cellproduct(s) combined with other tooth components may qualify as toothstem cell donors. Other mammalian patients and related donors havesimilar tooth derived stem cell product compatibility.

Referring to FIG. 3, as an example, harvesting stem cells from teethusually follows extraction of the entire tooth from an oral cavity.After the tooth 100 is removed, each tooth surface is cleaned and acircumferential cut is made through any enamel 116 and dentin 118proximate the crown 110 of the tooth, thereby allowing the crown 10 tobe separated from the root 114. Cutting through the enamel 116 anddentin 118, followed by removal of the crown 110, is operative to exposethe pulp cavity 120 and molar pulp and stem cells 124 contained withinthe pulp cavity. Alternatively, the molar pulp and stem cells 124 may beenucleated from the hard or solid portions of the tooth, such as thethird molar.

Referencing FIG. 1, the resulting molar pulp and stem cells 124 removedfrom the pulp cavity 120 are processed for future use. In one exemplarysequence, after the molar pulp and stem cells 124 are removed, thismixture is subsequently added to a solution of 3 mg/ml collagenase typeI and 4 mg/ml dispase for approximately 60 minutes at an ambient humanbody temperature of 98.6° F. The resulting product is a slurry, which isstrained to remove the stem cells 136 from solution, with the separatedstem cells being available for immediate use or available for cryogenicpreservation. The collagen portion 134 is also separated (and expandedif necessary) from the resulting slurry and may be used to create amixture 146 of collagen and stem cells for soft tissue applications.Alternatively, the molar pulp and stem cells 124, which includes theconnective pulp tissue, is diced without collagenase digestion forimmediate use or cryogenically banked.

In one exemplary variation, the stem cells 136, connective tissues 134,and tooth 110 are cryogenically banked using human leukocyte antigen(HLA) typing for future autologous and/or therapeutic uses. Dental pulpstem cells, if used in bulk without duplication or cell passes, mayrequire HLA typing for allogenic use. It is to be understood, however,that HLA typing need not be required for autologous and therapeutic usesin circumstances where extracted stem cells comprise a pure mesenchymalisolate. For example, a pure mesenchymal isolate from the dental pulphas no immunogenicity and may be included with ground or otherwiseprocessed tooth from an un-HLA matched allograft host source. Simpleblood typing (e.g. A, B, AB, O) is adequate for many mammals.

Those skilled in the art will recognize that use of a single autologousdonor for the stem cells and the bone and soft tissue graft source hasthe advantage of reducing or eliminating the risk of the mammalian bodyrejecting the medical implant and therapeutic stem cell compositions. Ofcourse, it is also within the scope of the disclosure to utilizemultiple donors. Where multiple donors are used as the source of eitherthe stem cells or the bone graft material (such as a ground tooth) orthe soft tissue material, these donors and patients can be matched usingstandard known matching means, such as, without limitation, HLA typing.HLA are proteins, or markers, found in most cells of a mammalian bodyand used to differentiate between native and foreign cells.

A close match between a patient's HLA markers and those of one or moredonors reduces the risk that the patient's immune cells will attack thedonor's cells. Moreover, a close HLA match improves the chances forgraft incorporation and vascularization. Engraftment occurs when thedonated cells start to grow and make new blood cells, which reduces therisk of a post-transplant complication called graft-versus-host disease(GVHD). GVHD occurs when the immune cells from the donated graft attackthe host body. If a patient is in need of an allogenic implant (whichuses cells from a family member, unrelated donor or cord blood unit), adoctor simply takes a blood sample to test the patient and donor for HLAtype. When a donor tooth and attached cells are harvested, blood can bedrawn and sent for HLA typing to make possible allogenic fresh orcyropreservation grafting.

Those skilled in the art are familiar with sterilization preservationtechniques for stem cells. The dental pulp stem cells, if enucleated(such as from a harvested, unerupted third molar), can have the surfacesterilized with antiseptic solutions (alcohol, betadine, etc). Cell lossto certain depths occurs depending on the solution chosen and the lengthof time exposed to the antiseptic. Many commercially available productscan sterilize the harvested stem cells and connective tissue forbacterial, viral and even for mycoplasm contamination (e.g., BIOMYC-2 isbased on minocycline, which is a tetracycline derivative; BIOMYC-3 isbased on the ciprofloxacin antibiotic, which is a member of thefluoroquinolone group). As discussed briefly beforehand, the dental pulpmay also be minced and a separation (e.g., collagenase) process carriedout to divide the pulp into stem cells and connective tissue prior tostorage. Alternatively, a tooth or teeth may be decontaminated en masswith stem cell containing pulp attached and subsequently cryopreservedintact. In any event, different sterilization methods may be requiredfor each of these different protocols. Also, oral decontamination (e.g.,Chlorhexidine) and other antiseptic techniques used during molarharvesting are operative to decrease the extent of bacterialcontamination and reduce the sterilization required, which in turnlessens the loss of stem cells in the processing and storage phases.

Stem cells harvested fresh will, after decontamination, need to betemporarily or long-term preserved to maintain stem cell viability.Those skilled in the art are familiar with preservation techniques forstem cells. Temporary preservation after initial sterilization can bedone with various solutions depending on the time before use or storage.Aedesta Cryopreservation Medium was originally developed for organpreservation by Lifeblood Medical, Inc., as Lifor, and optimized by CellPreservation Solutions, LLC. Other solutions have isotonic concentrationto closely match the human body. Phosphate buffer saline (PBS) is abuffer solution commonly used in biochemistry and has many uses becauseit is isotonic and non-toxic to cells. PBS is a salty solutioncontaining sodium chloride, sodium phosphate, and potassium phosphate.In addition to the correct choice of solution, depending on the time touse, various amounts of cooling and refrigeration may also be employedto insure maximum stem cell viability. This includes long-termpreservation and cryopreservation. See Optimized cryopreservation methodfor human dental pulp-derived stem cells and their tissues of origin forbanking and clinical use, Woods E J, et al., Cryobiology, 2009 October;59(2):150-7, the contents of which are incorporated herein by reference.

Those skilled in the art are also familiar with in vitro multiplicationof stem cells. European Patent EP1557461 details the ability toproliferate or establish undifferentiated pluripotent stem cells thatretain their differentiation potency by culturing pluripotent stem cellsin a medium free of a feeder cell, or a serum. Serum media are availablefrom various manufacturers specializing in marrow mesenchymal stem cell(MSC) passage (e.g. IVGN has recently released a reduced serum mediumformulation MesenPRO RS (IVGN Cat. #12746012)) optimized for theexpansion of human MSCs through multiple passages while maintainingtheir ability to differentiate into chondrogenic, osteogenic andadipogenic lineages. The aim is attained by using a culture medium forpluripotent stem cells comprising the known ingredients, which issupplemented with an inhibitor of an adenylate cyclase activity.

An exemplary technique for expanding cryopreserved stem cells(http://ink.primate.wisc.edu/˜thomson/protocol.html#thawing) involvesfirst removing the molar pulp stem cells from a liquid nitrogen storagetank. The cryovial is gently swirling in water bath until only a smallice pellet remains. Then the cryovial is completely submerged in 95%ethanol for final thawing. The cells are then gently pipetted from thevial into a conical centrifuge tube. Media is slowly added drop wise toreduce osmotic shock. While adding media, the cells are gently mixed inthe tube by gently tapping the tube (with a finger). Then, the cells arecentrifuged for a predetermined time, with an optional intermediateresuspension phase, followed by re-centrifugation. The cells are thenresuspended in 2 mL and add 0.5 mL per well of a 4 well plate that hasMEFs already plated on it. The media is changed daily. The cells arethen expanded on Matrigel-coated plates (kept cold throughout theprocess). The human molar stem cells grow until the colonies are largeand the cells are piled up ready for splitting. The cells can then bespit on Matrigel. Matrigel Aliquoting and plated stem cells need to befed everyday with a standard hES media. Differentiated cells should bepicked off of the plate if more than 5% of the culture isdifferentiated.

It should be noted that the tooth from which the stem cells areextracted might also be preserved. After the stem cells are extractedfrom the tooth, the tooth is bathed in an antiseptic solution, followedby a deproteinizing bathing in an alkali solution (1% concentration ofsodium hypochloride) for several hours. The enamel and dentin can beseparated sharply by various cutting methods. Alternatively, a hightemperature baking process (850° C. for 2-3 hours) follows extraction ofthe tooth from the alkali solution to calcinate the tooth. At hightemperatures, the dentin matter and enamel matter separate easily andprovide a separation approximately 60% dentin and 40% enamel. This hightemperature baking process is also operative to sterilize the tooth andprovide human hydroxyapatite free from infectious processes that havepreviously hindered utilization of human hydroxyapatite, such ashepatitis B.

For example, calcinated sheep teeth have been used a bone graftmaterial. See, F. N. Oktar, et al “Histopatological evaluation of toothderived hydroxyapatite and plaster of Paris as grafting material inrabbits,” Proceedings of the National Biomedical Engineering Symp. ofBiyomut 97, pp. 54-61, 1997, the disclosure of which is incorporatedherein by reference. Calcinated teeth have also been used as a humanbone-derived hydroxyapatite (HHA) plasma-spraying powder. In a similarexemplary application, powderized autologous THA could be plasma-sprayedonto an implant ingrowth interface and then autologous stem cells orslurry applied to optimize bone ingrowth. The starting powder is derivedusing the calcinations method as described in the following references,EP0489728 and WO9001955, the disclosures of each of which areincorporated herein by reference.

It should be noted that the tooth from which the stem cells areextracted might also be preserved in addition to, or independent of, thestem cells. The donor tooth may be preserved in whole (which includesthe two primary parts comprising the removed crown 110 and lower root114 (see FIG. 1)) or segmented into smaller pieces, such as by grinding.Also, deciduous teeth shed during the normal dental cycle could also besaved and cryopreserved. Regardless of how the residual tooth is to bepreserved, the tooth is initially bathed in an antiseptic solution.Further processing may take place after bathing the tooth in theantiseptic solution. For example, the tooth may be bathed in an alkalisolution for several hours, followed by a baking process to calcinatethe tooth, as discussed above, so that the enamel and dentin may beseparated and preserved separately. Alternatively, the hard componentsof the tooth may be dried after withdrawal from the aseptic solution andpreserved at room temperature in a sealed container.

Prior to, or subsequent to, tooth preservation, the tooth may be groundto create tooth particles of various size ranges. For example, dentinmay be ground to have particle sizes ranging between 100-150 μm. By wayof example, and not limitation, the molar tooth may be ground to havemean particle sizes in the range of 5-100 μm, 100-500 μm, and 500-1,000μm. Referencing FIG. 12, bone graft slurries 1204 for sub-cutaneousneedle guided injection 1206 into highly contained areas (vertebral bodyfracture, see FIG. 12A) generally have a 5-100 μm particulate size.Known milling and other techniques exist to sterilely process hardtissues, such as bone and teeth, to various dimensions (5-1000 μm) (see,e.g., U.S. Pat. Nos. 6,824,087 and 6,287,312, the disclosure of each ofwhich is incorporated herein by reference). These devices and other moreconventional non-medical milling devices (see, e.g.,http://www.alpinehosokawa.com) are adaptable to grinding a tooth intoparticle size ranges desired for various applications.

An exemplary method of the present disclosure includes combining dentalpulp stem cells collected from a tooth of a mammalian donor with nativetissue to form a stem cell graft product. This native tissue maycomprise tissue from the patient recipient, such as a tooth, or maycomprise tissue from the stem cell donor, such as the stem cell donor'stooth. In addition, the patient's own autograft tissues, synthetictissue substitutes (e.g. SIS, DePuy Orthopedics), or an HLAmatched/unmatched allograft may be utilized to expand the quantity ofgraft material for a particular application. Of course, any of a varietyof known methods of making a particulate or structural allograft orautograft may be used with the present disclosure. These include, forexample, the methods disclosed in U.S. Pat. Nos. 6,511,509 and7,018,412, the disclosure of each of which is incorporated herein byreference.

While one may use any human bone in lieu of a tooth for the particulatematerial according to the present disclosure, there are severalpotential advantages of using a tooth or teeth as the primaryparticulate material. Teeth are available for preservation fromchildhood to teenager providing a very large potential source of THA.Harvesting autograft bone and stems cells from different surgical sitesrequire additional surgery and donor site morbidity (such asinflammation, infection, and chronic pain that occasionally outlasts thepain of the original surgical procedure). As briefly discussedpreviously, the tooth calcination process creates a sterile product thatis non-infectious and that will not illicit a foreign-body reaction inhost tissues (HLA typing not necessary once tooth deproteanated).Consequently, the advantages of using bone and/or teeth of the patientinclude lowering the risk of rejection, abundant supply of stem cellsfrom unerupted teeth, and low to no risk of cancer or other diseases inthe stem cells or hard tooth.

In a circumstance where the stem cell donor donated four third molars,and where the matrix/scaffold is not insignificant in size, each of thethird molars is preferably ground to provide a greater volume of bonegrowth slurry to accommodate the larger matrix/scaffold. For example, iffour donor third molars are ground, it may provide enough matrixmaterial (including the porosity) for a 3-D cleft palate defectreplacement. Alternatively, or in addition, HLA matched or unmatchedteeth particulate may be utilized in place of the host ground teeth.Alternatively, or in addition, bone substitutes (HA, TCP, calciumphosphate, etc.) may be used alone or in combination with the hostground teeth to fabricate a larger vertebral body replacement or otherbone defect matrix. Moreover, the ground teeth may be used alone or incombination with the native tissue from the stem cell donor, thepatient, and/or an HLA matched/unmatched donor to construct the matrix.

Referring back to FIG. 1, in accordance with the instant disclosure, theground tooth 138 may be combined with the extracted stem cells 136 tocreate a bone graft slurry 148. Alternatively, the collagen and othersoft tissue 134 extracted from the tooth 138 may be combined with theextracted stem cells 136 to create a soft tissue slurry 146. Because ofthe numbers of stem cells 136 available, it may be that only a portionof the total harvested or cryopreserved cells are needed with theremainder being preserved for later medical use. In general, the largerthe applications (such as, multilevel spine fusion in scoliosis)requires more cells, while the smaller usages (such as, injection into ascaphoid non-union) require fewer cells. Also the ratio of stem cells136 to ground tooth 138 or collagen 134 may vary for example when toothderived collagen matrix 142 or a ground tooth particulate matrix 144 isto be seeded by immersion (i.e., soaked) in a liquid milieus of cellsthat diffuse into and attach onto the scaffold porosity 150, 152. Forinstance, the ratio of stem cells 136 to ground tooth particulate 138 issmaller for injection into a particulate matrix 144 with relativelylarger porosity. Likewise, the ratio of stem cells 136 to collagen 134is smaller for injection into a collagen matrix 142 with relativelylarger porosity. Conversely, the ratio of stem cells 136 to ground toothparticulate 138 is greater when the surface of the particulate matrix144 is grafted to allow for improved graft host junction incorporationor to allow the stem cell slurry 148 to flow into the microporsity orinterconnective channels. Similarly, the ratio of stem cells 136 tocollagen soft tissue 134 is greater when the surface of the collagenmatrix 142 is grafted to allow for improved graft host junctionincorporation or to allow the stem slurry 146 to flow into microporsityor interconnective channels. Further, an atrophic non-union may requirean increased ratio of stems cells to ground tooth. Nevertheless, thepreferred range of ratios of stem cells to ground tooth will dependgreatly on the anticipated use and graft needs.

In the same manner, the particulate size required for a particular bonegraft slurry 148 varies depending upon application. Similarly, thepreferred particle size(s) of the tooth 138 depends in part upon theporosity of the matrix or native host into or onto which the bone graftslurry is applied. Smaller particulate and lower viscosity bone graftslurry 148 and soft tissue graft slurry 146 are needed to penetrate moredeeply into the porosity of solid free formed scaffolds 142, 144.However, those applications where it is preferred for the bone graftslurry to stay localized with less containment available generally havea larger particulate size of 500-1,000 μm. A non-union fracture site orfilling a larger defect are usages that generally require this largerparticulate size. The intermediate sizes of ground tooth, 100-500 μm,are appropriate for a cementless implant ingrowth surface or a scaffoldwith larger porosity. Autologous and synthetic scaffolding are selectedfor optimal porosity depending upon the desired application.

As used herein, the matrix 144 into or onto which the bone graft slurryis applied is sometimes referred to as an implant scaffold. The speed ofresorption for a bone substitute depends in part upon the presence ofinterconnected macropores of adequate size (over 100 microns) in thematrix or native host allowing colonization of the implant byosteoclasts and then the resorption or biodegradation.

Referencing FIGS. 1 and 2, THA particles 200 of varying sizes and shapesmay also be manufactured from the ground teeth 138. THA particles 200tend to be larger in size to fill larger graft sites (e.g., acetabularosteolytic defects, segmental bone fragment loss in open fractures,spine fusions, etc.), while smaller in size when filling small defects(e.g., scaphoid non-unions—see FIG. 10). Larger THA particles 200 willalso be more frequently utilized in association with open procedures(see, e.g., FIGS. 5, 6, 12C), thereby causing the bone graft 148 to staymore localized. Size and shape in addition to overall porositypercentage and macropore size affect mechanical properties such ascompressive strength and crushability. THA particle 200 sizes will varyto meet these graft site mechanical, as well as, biologic demands.

THA particles 200 have certain porosities to promote the desiredstrength (or crushability) osteointegration and rate of incorporation orresorption. These particles 200 are often characterized by a porositypercent and a size percent of the characteristic macroporosity. A largermatrix/scaffold 144 will also have a greater pore interconnectivitypercent (the greater the percent the more likely that resorption,incorporation or revascularization are enhanced). Structural needs oftendictate the porosity as increasing porosity usually decreases thestructural integrity because porosity is a physical parameter thatvaries inversely with graft compressive strength. For instance, ifmechanical and structural properties of the matrix 144 are similar tothose exhibited by GRAFTYS® HBS cement, approximately 65-70 percenttotal porosity with about 8 percent macroporosity (pores from 100 to 300microns) would be preferred.

In concert with porosity considerations, the viscosity of the bone graftslurry 148 and soft tissue graft slurry 146 are also important. Forexample, if the viscosity is too high, the slurry may not adequatelypenetrate the pores of the scaffold 142, 144. Conversely, if theviscosity is too low, the slurry 146, 148 may prematurely migrate out ofthe pores of the scaffold 142, 144. If the viscosity of the bone graftslurry 148 or soft tissue graft slurry 146 carrier is too low, a carriermay be used to contain the slurry and scaffold, thereby lessening theimpact of viscosity on the bone or soft tissue ingrowth or regeneration.

An exemplary carrier for use with the molar stem cell slurries 146, 148of the present disclosure is Graftys HBS (www.graftys.com) (“Graftys”).Graftys is a cement material comprising a solid powder phase thatinitially forms a plastic paste by mixing with a liquid phase. Thisviscous paste transforms into a stiff paste during setting, withporosity increasing its mechanical strength progressively up tosaturation/hardening. In accordance with the instant disclosure, themolar stem cell slurry 146, 148 is optionally applied to a matrix 142,144 and implanted in contact with a site where bone/soft tissue growthor regrowth is desired. In order to reduce the impact of viscosity onthe molar stem cell slurries 146, 148, Graftys may be applied to coverthe matrix 142, 144 and bone growth/regrowth site as a cap. BecauseGraftys hardens into a containment shell relatively quickly, even bonegraft slurry with a relatively low viscosity is not a problem. By usingGraftys, it is possible to utilize relatively low viscosity slurry 146,148 to fully penetrate the matrix 142, 144, without the drawbacks ofthis same low viscosity slurry migrating out of the matrix hours afterimplantation. But if a carrier or cap is not utilized, additives may berequired to change the viscosity of the slurry 146, 148.

Viscosity increasing substances 154 for addition to graft slurries 146,148 include, without limitation, autogenous blood or blood products(platelet concentrates, plasma concentrates, etc). These substances maytake on a dual role to optimize viscosity, while at the same timeproviding biologic enhancement (growth factors, differentiation, etc).Platelet gels or autologous platelet gels increase the volume of cellsused in the graft slurry 146, 148 and also provide initial control ofbleeding (hemostasis) and reduce of post-operative bruising. In additionto providing initial control of bleeding (hemostasis) and platelet gelsrelease mediators to help modulate the inflammatory response and many ofthe cellular functions involved in wound healing. Much of these effectsare due to the presence of growth factors and cytokines within theplatelets, and the presence of an increased concentration of white bloodcells in the gel. Examples of growth factors include platelet derivedgrowth factor, insulin derived growth factor, and transforming growthfactor-beta among many others. Growth factors are proteins that impartspecific biochemical messages to specific target cells through specificmembrane receptors

Creation of platelet gels requires harvesting platelet-rich plasma (PRP)from whole blood and combining it with thrombin and calcium or otheractivators to form a coagulum. The whole blood may be taken from thestem cell patient/recipient or may be take from close relative or HLAmatching donor. This coagulum or “platelet gel” is mixed with the bonegrowth slurry, and possibly with growth factors and white cells, toprovide a therapeutic benefit when located in proximity to a surgicalwound. As would be expected, addition of the platelet gel to the graftslurry 146, 148 is operative to increase the viscosity of the slurry. Incertain circumstances, this may require further addition of a viscositylowering substance to the graft slurry 146, 148.

Viscosity lowering substances 155 for addition to graft slurries 146,148 include, without limitation, aqueous components (e.g., sterilewater) and solutions thereof that include one or more of the followingsubstances: sodium chloride, potassium chloride, sodium sulfate,potassium sulfate, ethylenediaminetetraacetic acid (EDTA), and phosphatebuffered saline solution. By way of example, 0.9% NaCl saline solution,available from Baxter International (www.baxter.com), may be added tothe graft slurry 146, 148 to reduce its viscosity.

While viscosity is an important consideration, other considerations maygive rise to incorporating other additives, such as antibacterial orantifungal additives 157, into the graft slurries 146, 148 and/ormatrix/scaffold 142, 144 to discourage infection at the tissuegrowth/regrowth location. The type and amounts of these additives 157will vary depending upon the potential pathogen (is often sitedependent) and the relative toxicity to the stem cells or boneinhibition effects. These additives 157 include standard antibioticmedicines and anti-microbial compositions such as, without limitation,penicillin and metal alloys of copper and silver. These additives 157also include standard antifungal medicines, including, withoutlimitation, econazole, fenticonazole, miconazole, sulconazole,tioconazole, amphotericin, nystatin, terbinafine, itraconazole,fluconazole, ketoconazole, and griselfulvin. In certain instances,antifungal medicines are combined with other medicines when two actionsare desired. For example, an antifungal medicine is often combined witha mild steroid, such as hydrocortisone, to treat fungal infections withconcomitant inflammation.

Additional additives 159 may be combined with (or incorporated into) thetissue graft slurry 146, 148 and/or matrix/scaffold 142, 144 such asgraft incorporation enhancing medicines and bone or collagenantiresorptive medicines. Bone-graft materials usually include one ormore components: an osteoconductive matrix, which supports the creationor ingrowth of new bone; osteoinductive proteins, which supportmitogenesis of undifferentiated cells; and osteogenic cells (osteoblastsor osteoblast precursors), which are capable of forming bone in theproper environment. The bone graft slurry 148 will provide the neededosteoinductive mineral via ground tooth 138, while the stem cells 136will provide the osteogenic progenitor cells. The combination andsimultaneous activity of many potential additives results in thecontrolled production and resorption of bone. These factors (residing inthe normal extracellular matrix of bone) include TGF-beta, insulin likegrowth factors I and II, PDGF, FGF, and BMPs.

Using current techniques, in vitro differentiation of mesenchymal stemcells toward the osteoblast lineage is possible. Stem cells are culturedin the presence of various additives such as dexamethasone, ascorbicacid, and b-glycerophosphate to direct the undifferentiated cell towardthe osteoblast lineage (see U.S. Patent App. Publication No.2009/0155216, which is incorporated herein by reference). The additionof TGF-beta and BMP-2, BMP-4, and BMP-7 to the culture media alsoinfluence the stem cells toward the osteogenic lineage. For example,marrow cells containing mesenchymal stem cells may be combined withporous ceramics and implanted into segmental defects, with bony growthoccurring as quickly as 2 months. Mesenchymal stem cells can also beseeded onto bioactive ceramics. Factor-based bone graft substitutes havebeen isolated and synthesized, used alone or in combination with othermaterials such as transforming growth factor-beta (TGF-beta),platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),and bone morphogenetic protein (BMP). The type and amounts of theseosteogenic enhancing/differentiating factors will vary depending uponthe site dependent needs.

Anti-resorptive medicines that slow or stop the natural process thatdissolves bone tissue, resulting in maintained bone density and strengthmay also be utilized in combination with the graft slurries 146, 148 ofthe instant disclosure. In circumstances where bone loss or density is aconcern, anti-resorptive medicines are operative to prevent or retardfurther development of osteoporosis. Exemplary anti-resorptive medicinesinclude, without limitation, bisphosphonates. Some examples ofbisphosphonates include alendronate, etidronate, ibandronate,risedronate, and zolendronic acid. Exemplary methods of coating animplant device with bisphosphonates are disclosed in U.S. PatentPublication No. 20060188542 and U.S. Pat. No. 7,163,690, the disclosureof each of which is incorporated herein by reference.

Autologous and Allograft Procedures

By way of example, and not limitation, the exemplary techniques andformulations of the present disclosure are useful in primary pediatricorthopedic procedures including spine fusion and other joint fusionssuch as ankle fusions for club feet treatment and fractures highnon-union rates such as scaphoid and distal third tibial fractures.Additionally, by way of example and not limitation, exemplary primarypediatric orthopedic procedures that can benefit from these products andmethods include, limb lengthening procedures, atrophic fracturenon-unions, and fractures with high non-union rates such as distal tibiafractures, scaphoid fractures, fifth metatarsal fractures, femoral neckfractures and clavicle fractures. Pediatric cases are ideally suited asthey may have molars available for harvest at the time of surgical need.Adult orthopedic uses mirror most of the above but also includecementless total joint replacement surgery, and other orthopedic bony,tendon and ligamentous conditions having a relatively high incidence offailure using prior art techniques and compositions of matter.

Spinal Fusion Example

Referring to FIGS. 1, 4 and 5, an exemplary procedure in the context ofprimary pediatric orthopedic procedures includes a spinal fusion. Such aprocedure necessarily involves harvesting dental pulp and isolatingautologous stem cells from an appropriate donor, whether from thepatient himself or an HLA matched relative. The harvesting may be doneimmediately prior to the spinal fusion procedure under the sameanesthetic or may be carried out well prior to the fusion procedurepresuming some sort of preservation technique is utilized for the stemcells, such as, without limitation, cryogenic preservation. In anyevent, it is preferred that within 72 hours of the primary pediatricorthopedic procedure, in this case a spinal fusion, the dental pulp stemcells 136 are combined with ground tooth particulate 138 to create astem cell molar graft slurry 148 for use in a pediatric spinal fusionprocedure.

By way of example, a pediatric spinal fusion procedure may be carriedout to correct deformities associated with scoliosis. Scoliosis spinalfusions are generally carried out using one of two approaches. A firstapproach is a posterior spinal fusion where a vertical incision in theposterior of the patient is made, proximate the spine, while the patientis lying on his/her stomach to expose the vertebrae. A second approachincludes making incisions on the lateral side of the patient, while thepatient is lying on his/her second lateral side, to access the spinalcolumn. During this second procedure, the patient's lung is deflated anda rib removed in order to reach the spine.

During surgery (either or both surgical exposures), after the spine isexposed, the surgeon attaches a metal plate 400 to each side of thepatient's spine 402 using hooks or screws 404 that are most commonlyattached to the vertebral bodies as is commonly known to those skilledin the art. Sublaminar wires (not shown) and screw fixation are utilizedat the multiple levels to be fused. The metal plates 400 attached to thespine ensure that the spine remains straight and rigid while the spinalfusion takes place.

Molar graft bone particles 406 can be added if available from autologousor allogenous sources. Autologous harvested bone graft 418 (from thebone removed from the spine 402 and iliac crest 408) is ground andthereafter added to the fusion mass along with the stem cell bone graftslurry 412. The bone graft slurry 412 is injected, using a syringe 414,proximate locations where bone fusion is preferred. In addition, THAparticles other bone graft extenders 422 and autologous (e.g., plateletgels) and allogenous or synthetic (e.g., BMP) growth factors may beapplied as determined by the surgeon. The entire length of the deformityis generally grafted (anywhere up to 15 levels between T3-L5), but inthis exemplary depiction only the L3-L5 vertebrae being fused.

By way of example, prior to injecting the bone graft slurry 412, aplatelet gel is prepared by harvesting sixty cc's (two ounces) of thepatient's blood just prior to surgery. The blood is placed into aspecially designed tube, which is centrifuged for about 15 minutes. Thiscentrifuge process concentrates up to 80% of the natural healing factorsthat are in the blood. The platelet concentrate is placed sterily on thesurgical field in a 10 cc syringe (not shown). This is paired with a 1cc syringe of calcium and purified bovine thrombin in a double-barreledsyringe, much like an Epoxy gun (not shown). The contents of thesyringes are mixed when the products are sprayed into the wound tocreate a sprayed platelet gel.

Thereafter, the stem cell molar graft slurry 412 is applied using thesyringe 414 to deposit slurry droplets throughout the fusion levels,with particular attention likely given to the proximal and distal fusionjunctions where non-union rates are the greatest. The bone graft slurry412 facilitates bone growth and fusion of the vertebrae segments (inthis case L3-L5) as the fixation plates 400 and screws 404 temporarilystabilize the spine 402. The patient is often braced to promote furtherspinal fusion healing by decreasing the likelihood of segmental motionbetween the vertebral segments holding them immobile and straight. Afterinjecting the molar graft slurry 412, the perispinal and fascial closure(not shown) operate to hold the molar graft slurry 412 contained in theregions where fusion is needed.

In this example, the molar graft slurry 412 comprises approximately 5cc's of slurry with a drop or two being placed at each level wherefusion is desired. The volume of the molar graft slurry 412 may begreater if mixed with blood, platelet concentrate, and/or serum 154.Exemplary needle gauges for use in injecting the exemplary molar graftslurry 412 include, without limitation, 12 gauge to 21 gauge.

In circumstances where the fusion site is closed with soft tissue, themolar graft slurry 412 can be injected after initial grafting and finalwound closure. Hemostasis to prevent the molar graft slurryextravasation can be obtained by packing a combination of Gelfoam(available from Upjohn, of Kalamazoo, Mich.) and thrombin, or bone waxcan be applied to the raw osseous surfaces to stop the bleeding. Packingwith lap sponges also helps to control the bleeding. The stem cell molargraft slurry may also be injected into the site at the end of theprocedure (necessarily a less viscous slurry with smaller particulatesize) when a layered closure is utilized in order to insure containmentof the slurry. Autograft or allograft ligament or synthetic soft tissueclosure materials (e.g., Small Intestinal Submucosa RESTOREOrthobiologic Soft Tissue Implant, available from DePuy Orthopedics,Inc.) may also be used to help create a containment compartment for themolar graft slurry 412 to prevent its extravasation and possiblestimulation of heterotopic bone formation.

In this example, the particulate size has a bimodal distribution varyingbetween 5-100 μm for injection. However, it should be understood thatparticle sized outside of this range may be used in accordance with theinstant disclosure. As discussed above, the molar graft slurry 412 mayinclude blood or a generic platelet gel (e.g., DePuy Biologics SYMPHONY™II Platelet Concentrate System) or other aqueous solutions to obtain theappropriate viscosity. It should be understood that the ratio of thedifferent component amounts (e.g., stem cells, ground tooth, and otheroptional components) correspondingly affects the viscosity of theslurry. Depending on whether the graft is to be applied to the fusionsite alone, or in conjunction with expanders, also affects ratio of thevarious components of the molar graft slurry 412. Larger ground toothparticulate 406 sizes and more viscous solutions, like plateletconcentrates, generally work better when the molar graft slurry 412 isapplied directly into the fusion site (with or without other autograft,allograft, synthetic materials and expanders). Moreover, the graft sitemay be sealed to contain the molar graft slurry 412 by using sealingproducts that harden such as, without limitation, Graftys HBS(www.graftys.com).

Alternatively, or in addition, the molar graft slurry 412 may beinjected at postoperative intervals to facilitate spinal fusion, mostcommonly at the proximal and distal fusion points, particularly whenradiographic evidence of fusion is lacking. By way of example,postoperative injections may be delayed hours or days after homeostasisand swelling have stabilized. In cases where fusion failure issuspected, particularly at the critical proximal and distal fusionsegments, a molar graft slurry 412 with a higher stem cell to THA ratiowould most often be used.

The foregoing procedure may be adapted to treat other joint fusionprocedures at index procedure, or postoperatively to augment or treat afailed or failing fusion due to poor biologic conditions inherent tocertain cases (e.g., cervical fusions (interbody and lamina), subtalarand tibiotalar arthrodeses, metacarpal wrist carpal arthrodeses).Introduction of a molar graft slurry 412 in accordance with the instantdisclosure addresses both the biological and the mineral contentconsiderations for optimal bone growth.

Bony Non-Unions Example (and Fractures with High Non-Union Rates)

Referencing FIGS. 6-11, another exemplary orthopedic procedure for whichbone graft slurries in accordance with the instant disclosure may beutilized involves bony non-unions that are atrophic or hypotrophicresulting from fractures that fail to unite due to poor biologicconditions. Certain fractures have very high rates of not healing suchas, without limitation, cervical, distal tibial diaphyseal fractures,clavicle fractures, femoral neck fractures, scaphoid, fifth metatarsalfractures.

Referring to FIGS. 9-11, an exemplary fracture with a high non-unionrate is the scaphoid fracture of the wrist 900. In the undisplacedfracture, a stem cell molar graft slurry 902 formulated in accordancewith the instant disclosure is injected by fluoroscopic guidance intothe fracture zone 904 using a syringe 906. The stem cell molar graftslurry 902 is contained within the fracture hematoma or contained by thesurrounding carpal ligaments and soft tissue 908. An external fixator,cast or brace (not shown) may also be used to hold the wrist carpal bonestable to limit motion and promote fracture healing. Normally, 3-6 monthof cast or fixator immobilization is required for healing. Increasedrate of healing or decreased rate of fracture non-union are extremelyvaluable from a cost and patient morbidity perspective. In a similarclosed percutaneous fashion, other non-displaced fractures known to havehigh non-union rates may be similarly treated. In addition, growthfactors and other healing agents may be added or formulated by thesurgeon as needed to create a stem cell molar graft slurry 902 promotingsuccessful or expedited healing.

When open reduction and internal fixation is required for the displacedscaphoid fractures, a Herbert Screw 910 is often used for fracturefragment fixation and stabilization. Stem cell molar graft slurry 902 isadded to the open fracture zone 904 with additional molar particle graftor autograft, as necessary. Drops of low viscosity stem cell molar graftslurry 902 with small THA particulate are added and operative topenetrate the fracture zone 904 (see FIG. 10). Conversely, a moreviscous stem cell molar graft slurry 902 may be used for fracturesevidencing greater bone particulate (evidence of the bone being crushed)or bone loss at the fracture zone 904. The soft tissues 908 surroundingthe fracture zone 904 are closed over the region to contain the stemcell molar graft slurry 902. Growth factors or other bone healingadjuncts may be added to further promote successful healing over ashortened period of time. Those skilled in the art will understand thatvarious external fixation adjuncts may be used to retain the bones inalignment such as, without limitation, external braces.

An exemplary stem cell molar graft slurry 902 is fabricated inaccordance with the instant disclosure. For a young child or teenagerpatient, unerupted wisdom teeth (such as tooth 1732 in FIG. 17) may beextracted and thereafter the pulp from the pulp chamber 120 (where thestem cells are located) is separated from the dentin and the toothenamel from the crown 110 (see FIG. 1). Depending upon the durationbetween tooth extraction and the surgical procedure to repair the bonynon-union, the stem cells are preserved using either short term or longterm preservation techniques. In exemplary form, for a young child orteenager, one or more unerupted molars are extracted, the pulp isseparated from the remainder of the tooth mass, and just prior to thetime of injection, the stem cell molar graft slurry 902 is prepared on asterile side table. Alternatively, if the molar stem cells werepreviously cryopreserved, the stem cells are unfrozen the day of thesurgery and combined with ground tooth to form the stem cell molar graftslurry 902. For application in bony non-unions, the bone graft slurry isformulated to embody a more viscous consistency.

Referring to FIGS. 6-8, an exemplary surgical procedure to repair a bonynon-union between a first bone section 600 and a second bone section 602includes surgically excising and curettaging to create a cavity 604 atthe non-union location, with both bone sections 600, 602 providing goodsurrounding bleeding bone. The cavity 604 is then grafted with autograftor HLA matched stem cell molar graft slurry 902. The stem cell molargraft slurry 902 may or may not include additional patient autograft(such as patient THA particles 200 shown in FIG. 2) or synthetic bonemineral extenders, biologic graft, and autologous patient growthfactors. Those skilled in the art, particularly surgeons, willunderstand which aspects to mix based upon one or more of the following:the foregoing disclosure; real-time considerations in the operatingroom; patient-specific considerations; and, innate knowledge. Theformulated stem cell molar graft slurry 902 is thereafter injected intothe cavity 604 using a syringe 906 to substantially fill the cavity. Thetop of the cavity 604 is then closed with a sealant or cap 606 (e.g.Grafty's). In addition, soft tissue 608, such as periostium or fasciallayers, also offer options of deep coverage and containment of the stemcell molar graft slurry 902. Moreover, the stem cell molar graft slurry902 may be cryopreserved and injected into the non-union location daysor weeks after the initial non-union surgical procedure.

The non-union location can alternatively be minimally exposed through alimited incision in order to create entry into the fibrous scar tissue.The stem cell molar graft slurry 902 is then injected at the location ofthe non-union to promote continued bone ingrowth and healing. Thesurrounding fibrous non-union tissues are operative to maintain theslurry 902 in position. Nevertheless, the slurry 902 maintains isposition also by use of external stabilization, such as an externalfixator or brace, that is operative to limit bone fracture non-unionmotion, thereby enhancing healing.

In another exemplary form, the stem cell molar graft slurry 902 isinjected percutaneously (without open incision) using needle guidancetechniques, directly into fractured areas without direct exposure of thefractured areas. This is of particular benefit in the treatment ofvertebral compression fractures in combination with or without vertebralbody expansion technologies. This conservative closed treatment preventsa myriad of complications that occur after attempted open treatment(that further devascularize these fractures). The same holds true forusing the stem cell molar graft slurry 902 for fracture grafting incases of delayed healing or significant initial bone loss.

Referring to FIG. 12, an exemplary application in accordance with theinstant disclosure is the formation of vertebral bone for the orthopedictreatment of vertebral fractures in osteoporotic patients or theinjection of the bone molar graft slurry into an osteoporotic vertebralbody pre-fracture. Recent assessments place the number of annualvertebral fractures to exceed over 700,000 cases. Pursuant to theinstant disclosure, vertebral bone formation may be accomplished inmultiple ways including, without limitation, in vitro bone formation andin vivo bone formation.

An exemplary percutaneous treatment of a vertebral fracture of avertebral body 1200 includes approaching the vertebral body through thepedicle 1202 to provide a conduit for a stem cell molar graft slurryformulation 1204 to be injected, using a syringe 1206, into a defect orfracture zone 1208. In this exemplary application, the stem cell molargraft slurry 1204 is formulated to include THA combined with autologousharvested bone or synthetic graft materials that are operative toincrease the mineral content of the slurry. In patients with harbor bonea minimally invasive incision is made down to the vertebral body pedicle1212 and a corresponding hole 1210 created in the vertebral body throughwhich the stem cell molar graft slurry 1204 is injected and then sealedoff using Floseal or another similar hemostatic agent. In thealternative, the hole 1210 may be filled using a bone or synthetic plug(not shown).

In vivo vertebral body replacement may also be accomplished using anartificial shaped or natural matrix/scaffold (not shown) in the shape ofthe vertebral bone to be replaced. The matrix/scaffold is formedin-vitro with the desired porosity is utilized as a chassis for boneformation. In exemplary form, the shaped matrix/scaffold may comprise,without limitation, one or more of the following: collagen,demineralized bone, tricalcium phosphate, hydroxyapatite, corallinehydroxyapatite, calcium sulfate, bioactive glass (SiO₂) and carbonatedapatite (e.g., OsteoGraft [DENTSPLY Friadent CeraMed, Lakewood, Colo.],Norian SRS [Synthes, Inc, West Chester, Pa.], ProOsteon [Interpore CrossInternational, Irvine, Calif.], Osteoset [Wright Medical Technology,Inc, Arlington, Tenn.]). After the matrix/scaffold formed, it issubsequently embedded with stem cell molar graft slurry comprising amixture of stem cells and tooth particulate as previously describedherein. In order to thoroughly embed the stem cell molar graft slurrywithin the matrix/scaffold, soaking and/or vacuum infusion may becarried out on the matrix/scaffold. Either or both of these techniquesmay be carried out in an operating room just prior to implantation ofthe matrix/scaffold. One skilled in the art is knowledgeable as to theplethora of ways a graft slurry may be applied to a matrix/scaffold tointroduce the stem cells and ground tooth into the microporosity. Inlike part, the viscosity of the slurry will in large part depend (or atleast should depend) upon the porosity of the matrix/scaffold to whichthe stem cell molar graft slurry is to be applied. As discussedpreviously, larger porosity can accommodate relatively higher viscosityslurries, whereas smaller porosity should be matched with lowerviscosity slurries. In any event, the stem cell molar graft slurry isoperative to promote incorporation and revascularization.

In the case of pending pathologic fractures or severely osteoporoticpatient (see FIG. 12D), injection of the stem cell molar graft slurrytakes place in a non-operative setting. By way of example, after aradiation procedure is carried out for a pathologic fracture, the stemcell molar graft slurry is injected proximate the pending fracture inthe operative setting. Metastatic fractures or pending pathologicfractures before or after irradiation pose a great problem fororthopedic surgeons. The instant stem cell molar graft slurries providea solution to these previously unsolved circumstances because the slurryprovides both the biology (molar stem cells) and the mineral content(THA) to facilitate bone reformation. In addition, chemotherapeuticagents, antibiotics as well as additional bone healing agents and growthfactors could be added to the slurry.

In exemplary form, the pending fracture comprises a metastatic osseouslesion. By using fluoroscopic guidance as shown in FIG. 12D, the stemcell graft slurry is injected precisely into the proper location tostimulate bone growth potential in one or more areas where radiation haspreviously eviscerated native bone growth potential. Alternatively,after minimal pathologic lesion curettage and debridement, the stem cellmolar graft slurry can be injected and accordingly covered andcontained.

Limb Lengthening Example

In in vivo bone formation, in contrast to in vitro bone formation, thenatural bone of the patient may be fractured, non-united, or includesome other bone cavity into which bone formation is preferred. In thiscircumstance, a bone cavity or bone defect already exists or is enlargedto receive bone graft slurry formulated in accordance within the instantdisclosure. Preparation of the bone cavity may include reaming andsurgical placement of screws or other fastening devices to maintain theresidual bone(s) in position so that subsequent bone formation isoperative to at least partially fill the cavity. In addition toreceiving the bone graft slurry, the bone cavity may receive a naturalor artificial matrix/scaffold. An example where in vivo bone formationis necessary for success is limb lengthening procedures.

Referring to FIG. 13, exemplary Ilizarov and/or limb lengthening orreshaping procedures (e.g., distraction osteogenesis) benefit frominfusion of a stem cell molar graft slurry 1300 formulated in accordancewith the instant disclosure. By way of background, limb lengthening orreshaping procedures involve osteotomy and periodically (i.e., daily)stretching the bone slowly so that bone formation occurs between thebone gaps caused by the fracture as a means to increase the overalllength of the bone. In the case of a patient with a leg lengthdiscrepancy, for example, due to the tibia and fibula bones in one legbeing shorter than the other, an osteo-distraction surgical procedurebegins by mounting an external Ilizarov fixation device 1302 to theproximal and distal ends of the tibia 1304 and fibula 1306 with respectto the locations 1308 where the intercalary osteotomy is to be done.Thereafter, the bone cortex is circumferentially cut via a minimalincision with the rigid fixation device preventing displacement of thebones 1304, 1306.

At open surgical exposure, a high viscous stem cell molar graft slurry1300 is prepared in accordance with the present disclosure and isinjected at the time of the osteotomy into the canals of the bones 1304,1306, proximate the fracture, to promote an initial patient osteogenesisresponse. The slurry 1300, if mixed well in advance of the Ilizarovfixation device mounting procedure, is preferably stored at a lowtemperature to avoid stem cell viability loss. If very small osteotomyincisions are utilized, then the slurry 1300 can be injected using asyringe 1310 through the osteotomy incisions and into the locations 1308where the bones 1304, 1306 were fractured. In order to ensure that thestem cell molar graft slurry 1300 is retained proximate the fracture,coagulated blood, waxes, absorbable gelatins (such as Gelfoam, and thecompositions disclosed in U.S. Pat. No. 6,863,900, the disclosure ofwhich is incorporated herein by reference), clotting agents, and/orsealants (e.g., platelet gel) may be utilized to cap or seal off theslurry, thereby maintaining it in location of the osteotomy.

By using the stem cell molar graft slurry, it is possible to increasethe rate of lengthening of the bone because the slurry provides thebuilding blocks for more rapid bone formation. At the same time, usingthe stem cell molar graft slurry decreases the incidence of needing tostop bone lengthening as a result of poor healing at the fracture site.

After osteotomizing the bone, the body begins to repair the fracture byingrowth of bone into the gap created by the fracture. This boneregrowth occurs as the result of the patients' own bone healing factorsand neo-vascularity entering the osteotomized region in combination withthe stem cell molar graft slurry 1300. Over time, the external fixationdevice is adjusted to incrementally increase the spacing betweenportions of the fixation device mounted to opposite sides of the bonefracture rings attached to the fractured bones 1304, 1306. Thisadjustment causes the fractured bone ends to be slowly pulled inopposite directions from the osteotomy site to gradually lengthen thebones (in this case, a tibia and fibula) to the desired length.Depending upon the extent of bone regrowth, five or more adjustments maybe made in a 24-hour period to increase the bone length by onemillimeter or more. Over time, the incremental distractions result in aconsiderable lengthening of the limb over time.

Alternatively, or in addition, after a predetermined duration postosteotomy as the lengthening procedure continues to be carried out days,and possibly weeks, from the initial Ilizarov fixation device mountingprocedure, a less viscous slurry 1300 (i.e., a slurry having higher stemcell to particulate ratio as compared to the slurry prepared and appliedduring the Ilizarov fixation device mounting procedure) is injectedthrough the soft tissue down to the osteotomy locations 1308 so that thedeep soft tissues, clot, and scar surrounding the bone regrowthlocations 1308 are operative to contain the slurry. This less viscousslurry 1300 may be injected on a single occasion or on multipleoccasions proximate the osteotomy site. The additional influx ofpluripotent stem cells and bone graft material is operative to infusecells and compatible biologic materials that rapidly transform into bonetissue—more rapidly than the natural human body is capable of. Incircumstances of failing distraction or when more rapid to distractionis desired, the stem cell molar graft slurry 1300 is formulated toinclude even higher ratios of stem cells to THA (with or withoutbone-enhancing additives).

Those skilled in the art, in view of the instant disclosure, willrealize that the less viscous stem cell molar graft slurries 1300 may becombined with growth factors where the expanding bone is not highlyvascularized. Likewise, these growth factors may be injected separatefrom the molar graft slurry 1300 proximate the bone formation locations.

Cementless Total Knee and Other Partial and Total Joint ArthroplasyExample

Referring to FIGS. 14-16, those skilled in the art are familiar withtotal knee arthroplasty (TKA) procedures. In such a procedure, theproximal end of the tibia 1400 is resurfaced to receive a prosthetictibial insert 1402 (commonly identified as a tibial tray) and the distalend of the femur (not shown) is resurfaced to receive a prostheticfemoral component 1404 (commonly identified as a femoral condylarreplacement). Likewise, the patella may also be resurfaced to receive acementless patellar component 1412. In exemplary form, the prosthetictibial insert 1402 regularly includes a stem 1406 that is inserted intothe tibial intramedullary canal and may be retained within theintramedullary canal using a cement to concurrently bonding to thetibial insert 1402 to the tibia 1400. Alternatively, prosthetic tibialinserts 1402 have been marketed with a stem 1406 having a porous ormicro-porous surface adapted to allow bone ingrowth so the stem ismounted directly to the tibia 1400 without cement. Likewise, someprosthetic femoral components 1404 include an interior surface (which isadjacent to the femur when implanted) that is porous and adapted toallow bone ingrowth, thereby mounting the femur to the femoral component1404.

It has been found in recent years that bone ingrowth into poroussurfaces of these prosthetic components 1402, 1404, 1412 does not occursufficiently to retain the components in proper orientation, therebyrequiring revision surgeries to cement the components in a permanentposition. But stem cell molar graft slurries 1408 formulated pursuant tothe instant disclosure offer a solution for insufficient bone ingrowth,thereby obviating cementing prosthetic components and revisionssurgeries inherent with cemented prosthetic components.

In exemplar foam, a stem cell molar graft slurry 1408 in accordance withthe instant disclosure is created. Because many recipients of jointreplacement components, in this case, knee replacement components, areolder and no longer have teeth from which stem cells may be gathered,the instant disclosure nonetheless provides an alternative for thesepatients. HLA allogenic matching can be utilized to obtain molar stemcells stored by a cryogenic bank or storage facility. However,blood-related family members of the patient may have teeth yet to beremoved from which stem cells can be harvested or these same familymembers may have stem cells from their teeth already harvested and incryopreservation. By using stem cells from close blood-related familymembers, the risks associated with an adverse immune reaction aresignificantly reduced.

As previously mentioned, it is not just the harvested molar stem cellsthat are important, but also making use of the tooth/teeth as a bonegraft material in conjunction with these stem cells. That being said,autograft extenders from the patient undergoing the arthroplastyprocedure reduce the amount of donor tooth material necessary (say, oneor two molar teeth) to fabricate an appropriate stem cell molar graftslurry 1408. The preferred autograft extender that is readily availablein most arthroplasty procedures is the finely morselizing bone from thepatient resected joint surfaces which is removed as a normal part of thearthroplasy procedure. An exemplary aspect of the stem cell molar graftslurry 1408 is more highly concentrated with stem cells than THA becausemorselizing bone from the patient can supply most of the hydroxyapatiteneeded, thereby preserving donor THA for later autologous needs.

In exemplary form, a typical TKA procedure is carried out to reshape thesurfaces of the distal tibia 1400 and proximal femur to receive thetibial and femoral components 1402, 1404, which results in removal ofsome of the tibia and femur. Those skilled in the art are familiar withthe initial incisions and resurfacing necessary to prepare the tibia1400 and femur for the prosthetic components 1402, 1404 and accordingly,only for purposes to promote brevity, those aspects are not explained indetail. As discussed directly above, some of the bone removed from thefemur and tibia 1400 is retained (not discarded) in order to function asan autograft extender. When the tibia and femur are resurfaced and readyto receive the cementless prosthetic tibial insert 1402 and femoralcomponent 1404, the resurfaced areas of the femur and tibia, as well asthe tibial intercondylar channel, are coated with the stem cell molargraft slurry 1408 using a syringe 1410 or by hand. Moreover, the slurry1408 may be manually applied using a spreader or may be sprayed onto thetibia 1400 and femur prior to mounting of the prosthetic implants.Alternatively, or in addition, the stem cell molar graft slurry 1408 maybe applied to the porous bone ingrowth surfaces of the prostheticimplants 1402, 1404 to promote and accelerate bone ingrowth into theporous surfaces of the implants. When applied to the prosthetic implants1402, 1404, the stem cell molar graft slurry 1408 is applied to coat theingrowth surface at the time just prior to final impaction. It should beunderstood, however, that the slurry 1408 need not be 100% uniform orcompletely covering all of the resurfaced areas to the tibia 1400 andfemur, the porous surfaces of the implants 1402, 1404, or theintercondylar channel.

The press fit impaction, along with limited weight bearing or jointmotion for a predetermined period of time, is operative to hold theprosthetic components 1402, 1404 stable to with respect to the tibia andfemur to allow for enhanced integration and accelerated bone ingrowth.For TKA, this predetermined period of time when limited weight bearingand joint motion is permitted is not so long as to be detrimental to thefinal range of motion of the prosthetic joint. No longer than two weeksof restricted joint motion and weight bearing should be necessary.

It should be understood that the foregoing exemplary procedure isequally applicable to fixed and mobile bearing knee joint implants, aswell as posterior cruciate retaining knee implants with significantcam-to-post interaction. Also, the molar graft methods and productsdescribed herein for TKA could be used to enhance fixation of anypartial or total ingrowth arthroplasty including but not limited to hipreplacement, shoulder replacement, ankle replacement, elbow replacement,and disk and vertebral body replacement.

Reconstructive Plastic Surgery Example

In reconstructive plastic surgery, the surgeon is tasked with finding aman-made solution to significantly distorted physical features in anattempt to reduce the distortion and, in some cases, approximate anatural appearance. These significantly distorted physical features maybe the result of genetic defects, illness, or physical injury (such asan automobile accident or post surgical procedure). The exemplary stemcell molar graft slurries of the instant invention are useful in plasticsurgery to repair these distorted physical features.

An exemplary reconstructive plastic surgical procedure where stem cellmolar graft slurries includes reconstructive surgery to repair bonesafter tumor removal that has left a defect of a specific shape in thebone. In a circumstance where a small 3-D defect is present and a 3-Dcustom scaffold is necessary to improve the cosmetic result, a solidfree form (SFF) scaffold may be fabricated using CAD/CAM methodologiesto create the requisite 3-D shapes from ground tooth particulate.Exemplary techniques to fabricate the matrices include, withoutlimitation, selective laser sintering and 3-D printing. Both of theforegoing process involve thermal temperatures operative to destroyliving cells, however, as has been discussed previously, thematrix/scaffold formation occurs prior to introduction of the stem cellmolar graft slurry. However, after arising technology may allow forlayering methods may allow scaffolds to be incrementally built up fromprefabricated thin (0.25 mm-1 mm) layers, stacked upon one another toform the final 3-D structure that provide an opportunity forincrementally seeding each layer with the stem cell molar graft slurry,rather than an immersion technique for cells intercalation. In anyevent, the 3-D matrices have porosity characteristics tailored to theend application, which involves stem cell molar graft slurries ofvarying viscosities depending upon the intended use.

As discussed previously, formulation of the stem cell molar graft slurryincludes utilizing stem cells extracted or derived from teeth. Theviscosity of this resultant slurry may be impacted by adding autogenousblood or blood products such as platelet jells to increase theviscosity, or aqueous solutions to decrease the viscosity. For example,the same bone graft slurry may be divided to create two distinctslurries—one with a relatively high viscosity, and one with a relativelylow viscosity for use with reconstructive surgeries.

By way of example, the customized 3-D matrix is soaked in theappropriate viscosity and particulate sized stem cell molar graftslurry. The particles of THA are chosen or ground to be smaller than thematrix pore size to allow the needed penetration into the 3-D matrix.Alternatively, or in addition, other custom or non-custom THA orsynthetic scaffolds may be used. Each addition matrix is preferablysoaked or coated in a stem cell molar graft slurry of the appropriateviscosity.

During the reconstructive surgical procedure, the bone(s) subject torevision is targeted and one or more bone sections or portions areremoved to eliminate or reduce the deformity. Thereafter, the 3-D matrix(loaded with the stem cell molar graft slurry) is attached to the endsor exposed portions of the bone that remain and is affixed using knownfixation methods. Those skilled in the art of reconstructive surgery arevery familiar with such fixation methods. These fixation methodsinclude, without limitation, screw fixation, wire fixation, platefixation, rod fixation, a hardening non-biologic cement (e.g., Palacos,available from Zimmer USA), and a biologic hardening cement-like product(e.g., Grafty's). Additionally, for smaller bone defects, the stem cellmolar graft slurry may be utilized alone or in conjunction with otherautologous patient bone, such as from the iliac crest.

Soft tissue defects may be similarly addressed with custom or standardcollagen or connective tissue matrices or scaffolds with porosities toaccept a stem cell collagen graft slurry.

Orthodontic and Maxillofacial Procedure Example

Orthodontic and the frequent accompanying maxillofacial reconstructiveprocedures are additional procedures where utilization of stem cellmolar graft slurries formulated pursuant to the present disclosure areadvantageous. Since the impacted third molar 1732 from the patient maybe available for distraction, the principle concern is the formation ofcustom THA matrices using fresh harvested molars. However, the instantinvention also makes use of stem cell molar graft slurries for use withstandard, universal shaped matrices, where one of more of the patient'smolars are harvested at the beginning of the reconstructive surgicalprocedure and processed as the other parts of the reconstructiveprocedure are undertaken (see e.g., FIG. 17). As discussed previously,it is not necessary to extract one or more teeth proximate to thereconstructive procedure as previously extracted teeth can be harvestedand put in cryopreservation until the time each is necessary just priorto the reconstructive surgical procedure.

Exemplary grafts/matrices for use with facial reconstruction proceduresmay be fabricated by rapid manufacturing techniques or be prepared inbulk form to fill a particular defect. As will be discussed in moredetail below, the grafts/matrices may be soaked in the stem cell molargraft slurry prior to or during the surgical reconstruction procedurewhile the patient is under anesthetic.

Referring to FIG. 17, an exemplary facial reconstructive procedure willbe described that makes use of a molar stem cell graft slurry 1700. Inthis exemplary procedure, the patient is undergoing sinus lift anddistraction osteogenesis procedures. It should also be noted, however,that other maxillofacial reconstructive procedures may make use of themolar stem cell graft slurry 1700 including, without limitation, ridgeexpansions. In sum, any gap created in maxillofacial bone is amendablefor application of the molar stem cell graft slurry 1700 to facilitatebone formation proximate the bone where the slurry is located to addressnon-unions, fractures, ingrowth and other reconstructive bony proceduresof the facial bones.

An exemplary reconstructive procedure for a sinus lift includesfracturing the maxilla 1702 along a predetermined plane 1704 in order toseparate an upper portion 1706 of the maxilla from a lower portion 1708of the maxilla that retains the patient's teeth. In this exemplaryembodiment, the separation of the maxilla portions 1706, 1708 creates awedge-shaped cavity that is filled with a bulk allograft or 3-D matrix1710 to generally fill the cavity. In a circumstance where a 3-D matrixis utilized, the stem cell molar graft slurry 1700 is applied to thematrix so that the slurry at least partially fills the voids in thematrix in order to promote initial junction healing and eventualincorporation of the maxilla portions 1706, 1708. By way of example, thestem cell molar graft slurry 1700 is injected using a syringe 1712 intothe interstices and micro-porosity of the biologic or synthetic matrix1710. In addition, a more viscous stem cell molar graft slurry (notshown) is prepared and injected to occupy at least a portion of thejunction between the maxilla portions 1706, 1708 and the 3-D matrix1710. This latter, more viscous, bone graft slurry may be prepared withless fluid constituency, but includes the same or similar particulatesizes as used with the less viscous bone graft slurry. Alternatively,the latter, more viscous, bone graft slurry may be prepared withslightly larger particulate sizes in order to increase the ability ofthe bone graft flurry to remain in place. However, where a bulkallograft is utilized in lieu of the 3-D matrix, a less viscous stemcell molar graft slurry is used to at least partially fill the grafthost junctions and/or into the porous matrix to promote bonereconstitution/integration.

In exemplary form, Grafty's (not shown) is utilized to seal the stemcell molar graft slurry 1700 in position at the junction between thenative maxilla portions 1706, 1708 and the 3-D matrix or allograft 1710.Alternatively, Skeletal Repair System (SRS) (available from Norian,Cupertino, Calif.), is an injectable paste of inorganic calcium andphosphate that may be utilized to form a hard covering to encapsulatethe bone graft slurry 1700 and retain it in the proper position. Forreference purposes, SRS typically hardens in a matter of minutes andfauns a carbonated apatite of low crystallinity and small grain sizesimilar to that found in the mineral phase of bone. SRS is useful as abone-graft substitute to augment cast treatment or internal fixation ofimpacted metaphyseal fractures. It should also be noted that the stemcell molar graft slurry 1700 can be added to either Grafty's or SRS tofill the pores of these materials, thereby further enhancing thepotential of the patient's body to incorporate these materials as a bonesubstitute.

After the slurry 1700 and matrix/allograft 1710 are implanted andproperly positioned, a plate 1714 is mounted to the maxilla portions1706, 1708 and the allograft/matrix 1710 using fasteners, such assurgical screws 1716. The plate 1714 ensures that there are staticinterfaces between the maxilla portions 1706, 1708 and theallograft/matrix 1710, thereby enabling the stem cell molar graft slurry1700 to promote bone formation without dynamic shifting of theinterfaces.

Because the molar graft slurry 1700 is operative to facilitate boneformation in any gap created between bone, a distraction osteogenesisprocedure also uses the slurry. In exemplary from, the mandible 1720 isfractured to create a rear 1722 and a forward portion 1724. In thisexemplary procedure, the fracture is made vertically in between thefirst and second molar. However, those skilled in the art willunderstand that other lines of fracture may be chosen based upon theunique circumstances presented by patients' anatomies. It should benoted that prior to the mandible 1720 fracture, surgical plates 1726 aremounted to what will be the rear and forward portions 1722, 1724 usingsurgical screws 1728. These plates 1726, as will be discussed in moredetail below, ensure proper alignment of the mandible 1720 post fractureand also operate to create a vise that retains the matrix/allograftbetween the mandible portions 1722, 1724.

After the fracture of the mandible 1720 is accomplished, the rear andforward portions 1722, 1724 are separated from one another, therebycreating a cavity, to allow insertion of a 3-D matrix or allograft (notshown). Similarly to the sinus lift procedure discussed immediatelyabove, the 3-D matrix or allograft is inserted into the cavity and astem cell molar graft slurry formulated pursuant to the instantdisclosure is retained within the interstices of the matrix/allograft.In addition, autograft and bone graft extenders along with growthfactors and other healing agents can be added to enhance the osteotomyor distraction procedures to optimize desired healing and bony union.After the stem cell molar graft slurry and the matrix/allograft are inposition, a wire 1730 is connected to the plates 1726 mounted to bothmandible portions 1722, 1724 and tensioned on order to pull the platestoward one another and sandwich the matrix/allograft therebetween inorder to retain the matrix/allograft in a compression fit.Alternatively, or in addition, the surgeon may use a plate that isconcurrently mounted to the matrix/allograft and one or both of themandible portions 1722, 1724 to ensure proper alignment of the mandibleand static interfaces between the matrix/allograft and the mandibleportions 1722, 1724.

It should also be noted that the foregoing mandible lengtheningprocedure may be carried out using an Ilizarov device (not shown). Insuch a circumstance, the Ilizarov device is mounted to the mandible,followed by a calculated fracture of the mandible. The Ilizarov deviceis thereafter manipulated to lengthen the mandible as the bone forms atthe fracture location. In this procedure as well, the molar graft slurry1700 is injected proximate the fracture location at the time of theinitial fracture and subsequently at periodic intervals to speed boneformation at the fracture location and decrease the time necessary tolengthen the mandible.

Cleft Palate Example

Referring to FIG. 18-20, another exemplary use of a stem cell molargraft slurry formulated in accordance with the instant disclosure is inbone replacement cleft palate surgeries. Somewhat unique to cleft palatesurgeries and some facial surgeries in general is the fact that thesurgical procedure takes place in the same oral cavity as the primarysource for the stem cells and teeth comprising the bone graft slurry. Asdiscussed previously, aberrant and/or unerupted teeth may need to beremoved prior to or during the surgical reconstruction procedure.However, it should be noted that the exemplary cleft palatereconstruction procedure need not exclusively utilize the stem cellsand/or teeth from the patient.

A cleft palate is due to the failure of fusion of the maxillary andmedial nasal processes (formation of the primary palate). In most cases,a cleft lip is also present. Treatment procedures can vary with the ageof the patient, where a majority of maxillofacial procedures are carriedout on juveniles between the ages of 10-12 when growth is lessinfluential as deciduous teeth are replaced by permanent teeth, thussaving the juvenile from repeated corrective surgeries. But, often atwenty-year term of care for the child born with a cleft lip and palateis necessary.

Within the first two to three months after birth, surgery is performedto close the cleft lip. Then a multitude of honey surgeries are neededdepending on the defect. To repair the palate 1800, the boundaries ofthe palate cavity 1802 are cleaned and soft tissue removed to expose thebone. Thereafter, the cavity 1802 is filled with THA particles 1816 anda stem cell molar graft slurry 1804, formulated in accordance with theinstant invention, using a syringe 1806. One or more fastening plates1808 are mounted to the palate to retain the palate in position. Softtissue 1814 is grafted to cover the stem cell molar graft slurry 1804and correspondingly hold the slurry within the cavity 1802 postinjection. Alternatively, other autogenous tissue or small intestinalsubmucosa (SIS) or surrounding advancible tissues may be used to closeand contain the grafted region.

Though not always necessary, it is also within the scope of thedisclosure to implant a custom 3-D THA matrix or autograft or allograftmatrix fortified with stem cell molar graft slurry to at least partiallyoccupy the cavity 1802.

In other surgical procedures, an active orthopedic appliance is securedto the cleft segments 1810, 1812 and engineered to directly transportthem into proper alignment (e.g., Latham-type appliance). The Latham issurgically inserted by use of pins during the juvenile's fourth or fifthmonth. As is understood by those skilled in the art, a cavity existsbetween the palate segments. This cavity may be injected with the stemcell molar graft slurry at the time of appliance placement, with thesurrounding soft tissue being operative to adequately contain the slurryinjection. After, the Lantham is in place, the doctor, or parents, turna screw daily to bring the cleft segments 1801, 1812 together to assistwith future lip and/or palate repair. At any time prior to Lanthamremoval, the doctor may inject stem cell molar graft slurry into thecavity to facilitate bone formation and closing of the palate.

Formulating an exemplary stem cell molar graft slurry for use with cleftpalate reconstruction and other facial reconstruction procedurespreferably includes using an autologous, unfrozen, stem cell source. Inthis manner, the stem cell molar graft slurry is prepared the as soon aspossible before the planned surgery and kept refrigerated until use. Inan exemplary circumstance, the stem cells are harvested from the cleftpalate patient just prior to the cleft palate reconstructive surgicalprocedure under the same general anesthesia by the coordinated effortsof a surgical team composed of maxillofacial and orthodontic surgeons.This molar or tooth harvesting procedure provides fresh, unaltered stemcells without the need for cryopreservation, which can result in stemcell number loss or pluripotency loss if stem cell duplication isnecessary.

Additionally, it may be advantageous to combine the hydroxyapatite fromthe patient's teeth separate or along with autograft bone to fabricate3-D replacement graft scaffolds prior to implanting the 3-D scaffold,the stem cell molar graft slurry is applied by soaking the scaffold inthe slurry (if less viscous) or by applying a more viscous bone graftslurry to the exterior of the 3-D graft, where the stem cells (andpossibly other patient autologous growth factors) are absorbed by themicroporosity to facilitate bulk graft incorporation and vascularizationdue to the augmented biology. The fabricated 3-D graft, if utilized, ismounted to the native tissue (e.g., host bone) being spanned using astandard mini fragment AO fixation device (available from Synthes USA)titanium plate or with screws in circumstances where the host bone andthe 3-D graft have been predrilled.

As with the foregoing examples, it may be advantageous to utilize a bonehardening graft material, such as Graftys, in order to hold the slurryin place when the voids around the graft are to be injected and theslurry is to be maximally contained.

Cleft Palate Maxilla Widening Example

At about age five, on occasion, active orthodontics are necessary towiden and even protract the maxilla. The oral and maxillofacial surgeonthen should determine whether an alveolar cleft is present. If so, thealveolar cleft is typically closed, along with any residual oral nasalfistulas. In other words, for patients with a cleft palate and analveolar cleft, the surgical procedure involves partitioning the mouthand nose by grafting.

The oral and maxillofacial surgeon is desirous to provide a frameworkthat supports bone formation at the cleft site, thereby providing asubstrate for the eruption of the proximate teeth. Flaps are elevated,which permit direct closure of the nasal mucosa, and allow placement ofthe bone graft comprising an allograft/matrix and stem cell molar graftslurry to complete closure of the palate with a “water tight” layer oforal-attached mucosa. While various homografts and alloplasts arecurrently used for purpose, the material of choice is autologous bone,which may also be used with the stem cell bone graft slurry formulatedpursuant to the instant disclosure. Autologous bone may be harvestedfrom the ileum, calvaria, mandible, tibia, or rib, for example. Theforegoing surgical reconstruction of the nasal aperture provides supportfor the alar base and a solid foundation for future nasalreconstruction.

After grafting, the orthodontist develops proper arch form and monitorsthe eruption of teeth adjacent to the grafted cleft. Occasionally thecuspid requires surgical exposure and orthodontic traction, and fromtime to time, attached tissue grafting is indicted. Accordingly, thestem cell molar graft slurry of the instant disclosure, along withautologous bone and growth factors, may be used to address these needs.

Often, the cleft palate patient has a congenitally absent lateralincisor (i.e., a cleft dental gap). One approach is to allow the cuspidto erupt into its proper anatomic position while maintaining the lateraledentulous position. When growth is completed, an osseointegratedimplant can be placed. This technique helps maintain proper arch formand tooth mass, thus providing support for the overlying facial softtissues. Additionally, any horizontal maxillary deficiency that presentsitself can be treated in early adolescent with osteodistraction. Simplybut, stem cell molar graft slurries formulated in accordance with theinstant disclosure teamed with Illizarov techniques are useful incarrying out an osteodistraction. It should also be understood by thoseskilled in the art that, at the time of surgery, residual fistulas andadditional bone grafting may be performed.

Tooth Crown Example

An exemplary bone augmentation application for stem cell molar graftslurries formulated in accordance with the instant disclosure includespreparation of the jawbone for dental crowns. As is known to thoseskilled in the art, placement of dental crowns requires enough jawboneto support them. In a typical situation of a chronically lost singletooth missing for several years, there may not be enough bone to supportthe desired crown. Often, the patient does not have enough bone becauseof tooth loss from periodontal disease, injury or trauma, or adevelopmental defect. If the jawbone is too short (up and down), toonarrow (side to side), or both, a bone augmentation procedure isnecessary to add bone mass to the jawbone before dental implants can beplaced. Current bone graft procedures involve extracting bone from otherparts of the patient's body (chin, ramus, hip, or tibia) or implantingbone-like materials into the jawbone, and waiting for the graftedmaterial to fuse with existing jawbone over several months.

Preexisting procedures may continue to be used with stem cell molargraft slurry fabricated in accordance with the instant disclosure.Initially, a viscous stem cell bone graft slurry is injected proximatethe interface between the jawbone and the implanted materials tofacilitate bulk graft incorporation and vascularization. Over the courseof healing, less viscous stem cell molar graft slurries may be injectedproximate the graft to accelerate fusion.

Alternatively, stem cell molar graft slurries fabricated in accordancewith the instant disclosure provide an alternative to harvesting thepatient's bone. For example, if the patient has an unerupted thirdmolar, a family member that could donate such a molar, or access to anHLA-typed molar, this molar provides a ready source for stem cells andhydroxyapatite utilized to fabricate a 3-D scaffold for anchoring at therecipient site. Hydroxyapatite from the harvested/donated molar, alongwith autograft bone, may be utilized to fabricate a 3-D replacementgraft scaffold. Prior to implanting the 3-D scaffold, the stem cellmolar graft slurry formulated pursuant to the instant disclosure isapplied by soaking the scaffold in the slurry (if less viscous) or byapplying a more viscous bone graft slurry to the exterior of the 3-Dgraft, where the stem cells are absorbed by the microporosity.

To position the 3-D scaffold at the recipient site, the dentist firstdrills holes in the existing bone to cause bleeding. The 3-D scaffold isthereafter anchored to the jawbone using titanium screws in a similarway to present day harvested bulk bone graft procedure. Likewise, a stemcell molar graft slurry is fabricated, preferably using portions of thepatient's molar and stem cells. This stein cell molar graft slurry isthen injected into the scaffold, followed by covering the scaffold withslurry and a protective membrane (or other protective covering) overboth the scaffold and slurry in order to prevent the stem cells frommigrating away from the scaffold. As should be understood by thoseskilled in the art, the underlying bleeding of the jawbone helps thevascularization of this bone graft and delivers growth factors forhealing. In addition, the stem cell bone graft slurry may becryogenically preserved and later injected periodically at the graftingsite in order to accelerate fusion.

Ligament/Cartilage Example

Referring to FIG. 1, in addition to the foregoing examples where steincell molar graft slurries were fabricated for treatment of various bonegrowth issues, it should be understood that the instant disclosure alsocombines stem cells 136 harvested from teeth 100 with soft tissue 134also derived from harvested teeth to produce a stem cell collagen molargraft slurry 146. When combined, the stem cells 136 and soft tissue 134from the harvested tooth 100 create a stem cell soft tissue graft slurryfor use in treating ligamentous and cartilaginous loss.

The process for extracting soft tissue from teeth, as well asperiodontal ligament, is well known in the art (See Isolation,cultivation and characterization of stem cells in human periodontalligament, Molnár B, Kádár K, Király M, et al Fogory Sz., 2008 August;101(4):155-61, the disclosure of which is incorporated herein byreference). Accordingly, only for purposes of brevity, a detaileddiscussion of isolating soft tissue from teeth and the periodontalligament has been omitted.

A stem cell soft tissue graft slurry (stem cell collagen molar slurry)formulated in accordance with the instant disclosure is, when injectedin vivo, operative to form connective tissue for ligament and other softtissue injuries and pathologies. The consistency of the tissue/stem cellmixture will also vary with application and containment needs. A 3:1collagen to stem cell ratio may be desirable for severely frayed tendoninjuries or where the tendon ends are becoming separated. For asegmental replacement of a tendon defect by a molar derived collagenmatrix, however, the ratio may be 3:1 stem cells to collagen.

Referring to FIG. 21, an exemplary circumstance involves a surgicalprocedure to repair a partially torn Achilles tendon 2100. In such acircumstance, conservative treatment may be desired in order to returnthe patient to normal functionality as soon as possible. However, thebiology of healing must be enhanced to improve tendon healing andthereby decrease the disabling time course of the injury. To enhance thebiology of healing, a stem cell soft tissue graft slurry may be injectedproximate the Achilles tendon to promote tendon regrowth.

Referring to FIG. 22, in an alternate, more severe, circumstance wheretendon restructuring is necessary, a stein cell soft tissue graft slurry2102 may be injected subcutaneously via a syringe 2104 into the tendonto be restructured or in the peritendon region. The soft tissuessurrounding the tendon or peritendon region are operative to retain thestem cell soft tissue graft slurry 2102 in location. The tendon isappropriately braced to protect the healing tendon zone, but for ashortened period of time due to the enhanced biology stimulated by thestem cell collagen graft slurry 2102.

In formulating a stem cell soft tissue graft slurry in accordance withthe instant disclosure, the slurry may be supplemented with thepatient's autologous blood products or concentrates to provide certaingrowth factors (e.g., tumor necrosis factor (TNF) and interleukin (IL))to further stimulate the desired soft tissue healing.

In the alternative, the stem cell soft tissue graft slurry 2102 may beapplied to a cell free, tooth derived, collagen scaffold 2106 mounted tothe ends of the tendon dehiscence to repair the gap between the end ofthe torn Achilles tendon. Exemplary collagen scaffolds 2106 for use intendon repair may be tubular and sutured to the tendon at opposite ends.The scaffold 2106 is preferably injected with a low viscosity solutionof stem cell collagen molar graft slurry 2102 operative to penetrate theporosity of the scaffold. Alternatively, the scaffold 2106 may be soakedin the slurry 2102 prior to implantation, again to allow the slurry toadequately penetrate the interstices of the scaffold.

Arthritis Treatment Example

In a circumstance where cartilage restructuring is desired, a stem cellmolar collagen slurry formulated pursuant to the instant disclosure maybe injected directly into joints. In such a circumstance, the softtissue of the joint capsule is operative to retain the stem cell softtissue graft slurry in location. Alternatively, or at the same time, asoft tissue stem cell slurry may be injected into the joint capsule totreat arthritic and cartilaginous pathologies. The injection may becarried out by simple sterile injection via syringe and the time ofinjection is not necessarily critical. For instance, the injection intothe joint capsule may occur at the end of arthroscopic procedure, muchlike a current steroid injection. In addition, the injection may occurin conjunction with a steroid injection, and/or injection of plateletconcentrates, and/or appropriate growth factors to promote the desiredhealing.

Veterinary Applications

While the foregoing examples all related to a combination of human stemcells and either hard or soft tissues taken from human teeth, themethods and corresponding formulations are equally applicable to speciesother than humans. For example, the instant disclosure provides asolution for fracture fixation, fusion, reconstructive, prostheticingrowth, and other orthopedic applications in veterinary applicationsas well. The veterinary orthopedic and soft tissue applications mirrorthose of the human discussed above and otherwise. Likewise, theharvesting of one or more teeth from animals depends upon species andthe corresponding dental anatomy.

Soft tissue applications include ligamentous and other soft tissueapplications (reconstruction and healing) where the stem cells are usedin conjunction with soft connective tissue (from the molar or fromallogenic sources). As with human applications, injection of a stem cellsoft tissue slurry into an animal (non-human) joint cavity with orwithout additional autologous blood cells and plasma is operative toprovide enhanced healing.

In non-human mammalian molar teeth, the pulp is similarly located at thecenter, or core of the tooth and in the unerupted state this pulpcontains stem cells like in a human molar. After eruption of the molar,the pulp region includes connective tissue, nerves, and blood vesselsthat nourish the tooth. But, stem cells remain for a period of time inexfoliated molar teeth as they do in human exfoliated deciduous teeth.As in the human tooth, special cells in the pulp, called “odontoblasts”form dentin.

The majority of a mammalian tooth is made up of dentin, which surroundsthe pulp. Primary dentin is dentin that is formed before tooth eruption;secondary dentin is dentin that is continually formed throughout thelife of the tooth. As the secondary dentin forms, the pulp chamberreduces in size. The dentin of the crown is encased in enamel and thedentin of the root is covered by cementum. Dentin consists of 50-85%inorganic hydroxyapatite crystals, combined with organic matrix (mostlycollagen) and water. Intertubular dentin (primary structural component)is comprised of hydroxyapatite embedded in a collagen matrix.Peritubular dentin is a collagen free hypermineralized tubular wall.Dental tubules filled with odontoblasts form the interface between thedentin and the pulp.

The dense, hard external covering of mammal teeth is enamel, whichconsists of more than 95% of the mineral hydroxyapatite. A hallmark ofmammals is that the enamel characteristically, consists of a complex ofbumps (cusps) and ridges, which together increase the surface area ofthe tooth. Therefore, the ground tooth for use as a graft comprises, asa majority, hydroxyapatite with some collagen content. The alveolar boneforms the jaw and the sockets into which the roots of the teeth extend.The periodontal ligaments are a collection of connective tissue thathelps to hold the tooth in the socket. These ligaments attach to thecementum of the tooth and the alveolar bone. Simply put, the same humanharvesting preservation and uses are possible in most mammals. Becauseof some unique dental anatomy for certain species, variability for idealharvest timing (unerupted, premolar, shed teeth, etc), preservation anduses in animals of similar species and compatibility (ABO and or HLA)may be required.

Equine Examples

A foal typically will have a total of 16 teeth (four incisors or frontteeth, and 12 premolars or back teeth). At four to six weeks of age,four more incisors will erupt and at approximately six to nine months ofage the last set of up to 24 deciduous incisors will erupt. All of theseteeth are replaced during the time up to 5 years of age. Consequently,there is a constant eruption and loss of deciduous teeth, overlappingwith eruption of the permanent teeth during this period. Eventually upto 44 permanent teeth might be present in the horse's mouth—half ofthese are in the mandible and the other half are in the maxilla. Unlikedogs, cats, and humans, equine teeth continue to erupt throughout life.

At the same time, rudimentary premolar wolf teeth (referred to as “pml”)may erupt in front of the upper cheek teeth. These premolar wolf teethare small premolars which appear on the mandible above and usuallyslightly ahead of the molars. But in some horses, especially thestandard bred horse, it is not uncommon to also get wolf teeth in themaxilla (and often “blind” unerupted). These teeth are vestigial, thatis, they serve no purpose and may interfere with biting of the horse. Itis therefore advisable to remove wolf teeth while the horse is stillyoung as these teeth will eventually fuse with the bones of the skullmaking extraction far more difficult as the horse gets older. These wolfteeth, particularly the unerupted “blind” are an ideal source of molarpulp (and soft tissue containing stem cells) and hard tooth matter foruse in formulating exemplary stem cell molar grafts for bone and softtissue.

At twelve months of age, the first of the permanent cheek teeth begin toerupt, so it is a good time to ensure that normal eruption is occurringand to identify any other problems. From 1 year to 6 years of age horseswill shed their first set of 24 deciduous teeth and up to 44 permanentteeth will erupt through the gums. The structure of the chewing teeth(premolars and molars) in advanced horses is that the crowns areelongated relative to primitive mammals, so that they becomehigh-crowned (i.e., hypsodont). Highcrowned horse teeth have severallayers of folded enamel, forming “lakes” or fossettes on the chewingsurface. The extended period of crown growth is accomplished byregulation of the stem cells containing cervical loop. There are alsocontinuously growing teeth in which the cervical loop is maintainedthroughout the lifetime of the animal. The longer the stem cell niche ismaintained and differentiated progeny of the stems cells are produced,the higher the crown.

A multitude of opportunities exist to harvest tooth and stem cellmaterials from equine animals. For example, unerupted wolf teeth are anexcellent source of both tooth hydroxyapatite and stem cells. And thestem cell niche in the cervical loop of the highcrowned molars representa source of stem cells available at any time during the horse's life.

Since 2003, veterinarians have used autologous Human adipose derivedmesenchymal stem cells (ADMSCs) to treat tendon and ligament injuriesand joint disease in horses on a commercial basis (e.g., Vet Stem,BioScience, Ltd. UK). As these cells are classified as “minimallymanipulated,” these autologous stem cell therapies do not require FDAapproval. Studies and multiple anecdotal clinical experience demonstratethat autologous ADMSC therapy is of clinical benefit in horses withorthopedic conditions. ADMSC has also shown therapeutic success inequine tendonitis demonstrating statistically significant improvement ininflammatory cell infiltrate, collagen fiber uniformity, polarizedcollagen fiber crimping, overall tendon healing score, and collagenoligomeric matrix protein scores.

Equine Examples

Referring again to FIG. 23, successful equine fracture repair isdependent on the ability of the horse to generate enough new bone intime to stabilize the fracture before the onset of complications. Anexemplary use of a stem cell molar bone graft slurry 2300 withappropriate auotologous additive is shown in FIG. 23 being injected intoa non-displaced fracture of a lower leg bone 2302 of a horse 2304. Asdiscussed above, the stem cell molar bone graft slurry 2300 isformulated to include stems cells from one or more equine teeth inaddition to hard particulate matter derived from equine teeth.Preferably, both the stem cells and tooth particulate matter are takenfrom the equine to be treated with the stem cell molar bone graft slurry2300. However, close matches between equines may provide for withdrawalof stem cells from teeth of closely related animals and thereafterimplantation/injection of the stem cell molar bone graft slurry 2300without complications stemming from immune response and rejection of thestem cells and tooth particulate. Similarly, and not inclusively,reconstructive application, non-union treatments, fusions could all beapplications to one of normal skill in the area of equine orthodontic,and orthopedic bone procedures.

A further use of stem cells derived from an equine donor includes a stemcell molar soft tissue graft slurry 2306 formulated for injectionproximate a lower leg injured ligament 2308 of a horse 2304. Similarlyto ligamentous application discussed previously as to humans, the stemcells and soft tissue are extracted from a donor tooth and latercombined to create the soft tissue slurry 2306. Depending upon thefrequency of the injection and the site of injection, the slurry 2306may be modified to include growth factors and other native additives, inaddition to viscosity modifiers, in order to ensure the slurry isproperly retained in the region of interest. Stem cell molar soft tissueslurries 2306 derived from equine teeth and formulated in accordancewith the instant invention may be useful for the treatment ofligamentous injuries that plague horses, particularly those that arecommon to competitive jumping and racing horses.

In addition, an even further use of stem cells derived from an equinedonor includes a stem cell molar slurry 2310 formulated for injectionproximate an arthritic joint 2312 of the lower leg of a horse 2304.Similarly to joint injection applications discussed previously as tohumans, the stem cells and pertinent soft tissue extracts from an equinedonor tooth are utilized to create a stem cell slurry 2310. Dependingupon the frequency of the injection and the site of injection, theslurry 2310 may be modified to include growth factors and other nativeadditives, in addition to viscosity modifiers.

Canine Examples

Puppies have 28 teeth, while adult dogs have 42. The first deciduousteeth to come in are the canine teeth, followed by the incisors,premolars, and molars. Puppies generally start to lose the deciduousteeth at 2-3 months of age. Puppy teeth contain stem cells andhydroxyapatite from the puppy teeth may be harvested and stored as thepermanent deciduous teeth and canines erupt. But before the incisors,premolars, and molars erupt, in precursor form within the alveolar bone,the teeth may be harvested in a similar fashion to that described abovefor human teeth. In addition, when permanent and deciduous teeth arepresent at the same site, the deciduous tooth may be surgically removedto provide another source for tooth, soft tissue, and stem cell harvest.Consequently, hard tooth constituents, stem cells, and connectivetissues are preferably harvested at the time of the orthopedic and softtissue repair procedures. However, because of the opportunity thatexists when canines are young, the disclosure provides a means ofharvesting puppy teeth and cryogenically preserving these teeth so thatlater utilization of the stem cells, hard constituents, and soft tissueconstituents may be used in later life of the canine for one or moreorthopedic and soft tissue repair procedures.

Referring to FIG. 24, an exemplary use of a stem cell slurry 2400formulated in accordance with the instant disclosure for injectionproximate an arthritic hip joint 2402 of a canine 2404. Similarly tojoint injection applications discussed previously as to humans, the stemcells extracted from a canine donor tooth are utilized to create a stemcell bone slurry or stem cell soft tissue slurry containing a largepopulation of stem cells available for conversion into soft tissue, suchas the cartilage providing a bearing surface between the femur andacetabulum. Depending upon the frequency of the injection and the siteof injection, the slurry 2400 may be modified to include growth factorsand other native additives, in addition to viscosity modifiers.Likewise, the stem cell slurry 2400 may also be used to treat caninecartilage and soft tissue joint pathologies.

Another exemplary use of a canine stem cell slurry includes a stem cellcollagen slurry 2406 used to repair nerve injury where both stem cellprecursors and collagen and connective tissue material is needed fornerve regeneration. In exemplary from, the stem cell collagen slurry2406 is formulated from canine stem cells and the soft tissue extractedfrom one or more canine teeth. Preferably, the stem cells and softtissue are taken or have been taken from the canine patient. The stemcell collagen slurry 2406 is injected proximate the nerve damage 2408(in this case, a nerve tear) in order to promote nerve regeneration.

Alternatively, a canine stem cell collagen slurry may also be injectedproximate ligamentous and tendon injuries in order to promote ligamentand tendon repair. Those skilled in the art will readily understand thevarious applications for a stem cell collagen slurry based upon theplethora of injuries and degradations suffered by canines.

In accordance with the present disclosure, an exemplary use of a stemcell molar bone graft slurry (not shown) with appropriate auotologousadditive is to treat a non-displaced fracture of bone of a canine. Suchan exemplary stem cell molar bone graft slurry is formulated to includecanine stems cells from one or more canine teeth, in addition to hardparticulate matter derived from one or more danine teeth. Preferably,both the stem cells and tooth particulate matter are taken from thecanine to be treated with the stem cell molar bone graft slurry.However, close matches between canines may provide for withdrawal ofstem cells from teeth of closely related animals and thereafterimplantation/injection of the stem cell molar bone graft slurry withoutcomplications stemming from immune response and rejection of the stemcells and tooth particulate. Similarly, and not inclusively,reconstructive application, non-union treatments, fusions could all beapplications to one of normal skill in the area of canine orthodontic,and orthopedic bone procedures.

While the foregoing examples have been described with respect tocanines, it is also within the scope of the disclosure to harvest felineteeth and utilized one or more of the soft tissue, stem cells, and hardconstituents of a feline tooth to formulate a stem cell slurry. Thoseskilled in the art will be readily able to formulate a stem cell slurryfor a feline based upon the foregoing disclosure. Consequently, forpurposes of brevity, a more detailed explanation has been omitted.

Persons of skill in the art will understand that the foregoing methodsare also applicable to non-human mammals and reptiles, includingutilization of allograft bone sources and synthetic sources of scaffold.Moreover, the disclosure may be used to replace soft tissue, such ascollagen, ligament tendon and cartilage, in addition to or instead ofbone.

Following from the above description and disclosure summaries, it shouldbe apparent to those of ordinary skill in the art that, while themethods and apparatuses herein described constitute exemplaryembodiments of the present disclosure, the disclosure contained hereinis not limited to this precise embodiment and that changes may be madeto such embodiments without departing from the scope of the invention asdefined by the claims. Additionally, it is to be understood that theinvention is defined by the claims and it is not intended that anylimitations or elements describing the exemplary embodiments set forthherein are to be incorporated into the interpretation of any claimelement unless such limitation or element is explicitly stated.Likewise, it is to be understood that it is not necessary to meet any orall of the identified advantages or objects of the invention disclosedherein in order to fall within the scope of any claims, since theinvention is defined by the claims and since inherent and/or unforeseenadvantages of the present disclosure may exist even though they may nothave been explicitly discussed herein.

What is claimed is:
 1. A medical implant comprising: physicallydisrupted, non-enzyme-digested tooth pulp including a plurality ofnon-cultured stem cells; and a matrix.
 2. The medical implant of claim1, wherein the physically disrupted, non-enzyme-digested tooth pulp isselected from the group consisting of a diced tooth pulp, a minced toothpulp, and combinations thereof.
 3. The medical implant of claim 1,wherein the physically disrupted, non-enzyme-digested tooth pulp is asterilized, physically disrupted, non-enzyme-digested tooth pulp, andwherein the sterilized, physically disrupted, non-enzyme-digested toothpulp includes a plurality of stem cells that are living.
 4. The medicalimplant of claim 1, wherein the plurality of stem cells includepluripotent stem cells.
 5. The medical implant of claim 1 wherein thematrix is a biological matrix.
 6. The medical implant of claim 5 whereinthe biological matrix is chosen from the group consisting essentially ofa plurality of mammalian tooth particles, a plurality of bone particles,soft tissue, and combinations thereof.
 7. The medical implant of claim 6wherein the plurality of mammalian tooth particles is a plurality ofground mammalian tooth particles.
 8. The medical implant of claim 7,wherein the plurality of ground mammalian tooth particles includedentin, enamel, or mixtures thereof.
 9. The medical implant of claim 7,wherein the plurality of ground mammalian tooth particles includeparticles of tooth derived hydroxyapatite.
 10. The medical implant ofclaim 7, wherein the plurality of mammalian ground tooth particlesinclude one or more endogenous or exogenous growth factors.
 11. Themedical implant of claim 10, wherein the one or more endogenous orexogenous growth factors are chosen from transforming growth factorbeta, insulin like growth factor I, insulin like growth factor II,platelet-derived growth factor, fibroblast growth factor, and bonemorphogenetic proteins.
 12. The medical implant of claim 7, wherein theplurality of mammalian ground tooth particles have a mean particle sizein the range of 5 μm to 100 μm.
 13. The medical implant of claim 7,wherein the plurality of ground mammalian tooth particles have a meanparticle size in the range of 100 μm to 500 μm.
 14. The medical implantof claim 7, wherein the plurality of ground mammalian tooth particleshave a mean particle size in the range of 500 μm to 1,000 μm.
 15. Themedical implant of claim 5 wherein the biological matrix includes anautologous tissue.
 16. The medical implant of claim 1 wherein theimplant is shaped for implantation into a defect in a tissue.
 17. Themedical implant of claim 1 wherein the implant is shaped in the shape ofat least a portion of a bone.
 18. A medical implant comprising:physically disrupted non-enzyme-digested tooth pulp including aplurality of stem cells; and a matrix.
 19. The medical implant of claim18 wherein the matrix is chosen from the group consisting essentially ofa plurality of mammalian tooth particles, a plurality of bone particles,soft tissue, and combinations thereof.
 20. A medical implant comprising:physically disrupted, non-enzyme-digested tooth pulp including aplurality of stem cells.
 21. The medical implant of claim 20, whereinthe physically disrupted, non-enzyme-digested tooth pulp is selectedfrom the group consisting of a diced tooth pulp, a minced tooth pulp,and combinations thereof.
 22. The medical implant of claim 20, whereinthe physically disrupted, non-enzyme-digested tooth pulp is asterilized, physically disrupted, non-enzyme-digested tooth pulp, andwherein the sterilized, physically disrupted, non-enzyme-digested toothpulp includes a plurality of stem cells that are living.
 23. The medicalimplant of claim 20, wherein the plurality of stem cells includepluripotent stem cells.
 24. The medical implant of claim 20, wherein theimplant is shaped for implantation into a defect in a tissue.
 25. Themedical implant of claim 20, wherein the implant is shaped in the shapeof at least a portion of a bone.
 26. The medical implant of claim 20,wherein the stem cells are non-cultured stem cells.